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Neurotransmitter Transporters

Contemporary Neuroscience
Neurotransmitter Transporters: Structure,
Function, and Regulation,
2nd edition,
edited by Maarten E. A. Reith, 2002
The Neuronal Environment: Brain
Homeostasis in Health and Disease,
edited by Wolfgang Walz, 2002
Pathogenesis of Neurodegenerative Dis-
orders,edited by Mark P. Mattson,
2001
Stem Cells and CNS Development,
edited by Mahendra S. Rao, 2001
Neurobiology of Spinal Cord Injury,
edited byRobert G. Kalband
Stephen M. Strittmatter,2000
Cerebral Signal Transduction: From
First to Fourth Messengers,edited
byMaarten E. A. Reith, 2000
Central Nervous System Diseases: Inno-
vative Animal Models from Lab to
Clinic,edited by Dwaine F.
Emerich, Reginald L. Dean, III,
and Paul R. Sanberg,2000
Mitochondrial Inhibitors and Neurode-
generative Disorders,edited by
Paul R. Sanberg, Hitoo Nishino,
and Cesario V. Borlongan,2000
Cerebral Ischemia: Molecular and Cel-
lular Pathophysiology,edited by
Wolfgang Walz, 1999
Cell Transplantation for Neurological
Disorders,edited by Thomas B.
Freemanand Håkan Widner,1998
Gene Therapy for Neurological Disor-
ders and Brain Tumors,edited by E.
Antonio Chiocca and Xandra O.
Breakefield,1998
Highly Selective Neurotoxins: Basic and
Clinical Applications,edited by
Richard M. Kostrzewa,1998
Neuroinflammation: Mechanisms and
Management,edited by Paul L.
Wood,1998
Neuroprotective Signal Transduction,
edited by Mark P. Mattson,1998
Clinical Pharmacology of Cerebral
Ischemia,edited by Gert J. Ter
Horstand Jakob Korf,1997
Molecular Mechanisms of Dementia,
edited by Wilma Wasco and
Rudolph E. Tanzi,1997
Neurotransmitter Transporters: Struc-
ture, Function, and Regulation,
edited by Maarten E. A. Reith,
1997
Motor Activity and Movement Disorders:
Research Issues and Applications,
edited byPaul R. Sanberg, Klaus-
Peter Ossenkopp, and Martin
Kavaliers,1996
Neurotherapeutics: Emerging Strate-
gies,edited by Linda M. Pullan
and Jitendra Patel, 1996
Neuron–Glia Interrelations During
Phylogeny: II. Plasticity and Regen-
eration,edited byAntonia
Vernadakis and
Betty I. Roots, 1995
Neuron–Glia Interrelations During
Phylogeny: I. Phylogeny and Ontog-
eny of Glial Cells,edited byAntonia
Vernadakisand Betty I. Roots,
1995

Neurotransmitter
Transporters
Structure, Function,
and Regulation
Second Edition
Edited by
Maarten E. A. Reith
College of Medicine, University of Illinois, Peoria, IL
Springer Science+Business
Media, LLC

© 2002
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise without written permission from the Publisher.
All authored papers, comments, opinions, conclusions, or recommendations are those of the
author(s), and do not necessarily reflect the views of the publisher.
This publication is printed on acid-free paper.∞
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed
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Cover illustration:
Cover design by Patricia F. Cleary.
Production Editor: Mark J. Breaugh.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Neurotransmitter transporters: structure, function, and regulation / edited by Maarten
E. A. Reith.-- 2nd ed.
p. cm. — (Contemporary neuroscience)
Includes bibliographical references and index.
1. Neurotransmitters. 2. Carrier proteins. I. Reith, Maarten E. A. II. Series.
QP364.7 .N4757 2002
612.8'22—dc21 2001051651

Softcover reprint of the hardcover 2nd edition 2002
Springer Science+Business Media New York
Originally published by Humana Press Inc. in 2002
ISBN 978-1-61737-267-4 ISBN 978-1-59259-158-9 (eBook)
DOI 10.1007/978-1-59259-158-9

Preface
Neurotransmission is a multicomponent process. Transmitters,
released by neuronal activity, act on pre- and postsynaptic receptors,
and many books detail advances in the receptor field. In addition, after
their release from nerve endings, transmitters are removed from the
neuronal vicinity by uptake into neuronal or glial cells by specific trans-
porter proteins that have been studied intensely over the last 30 years;
this information is scattered throughout numerous publishing
vehicles. Therefore, the primary aim of this second edition of Neu-
rotransmitter Transporters: Structure, Function, and Regulation is to offer
a comprehensive picture of the characterization of neurotransmitter
transporters and their biological roles. The transporter field has moved
forward in stages. In the first phase, progress came from the use of
substrate or blocker ligands selectively targeting transporters, the
application of model systems allowing the study of transmitter trans-
port shielded from storage, and the development of mathematical
models for describing transport phenomena. In the second phase,
roughly covering the last decade, advances in DNA techniques
allowed the cloning of numerous genes coding for different transporter
proteins. In the current, third stage, a wealth of information is being
accumulated in studies relating transporter structure with function,
experiments addressing regulation by posttranslational transforma-
tion, investigations into transport modulation by trafficking processes
and genomic influences, characterization of channel properties of trans-
porters by electrophysiological approaches, and the creation of transgenic
animals under- or overexpressing a given transporter protein.
The first edition of Neurotransmitter Transporters: Structure, Function,
and Regulation was published at the brink of the above 2
nd
and 3
rd
phase;
the current, second edition finds the transporter field fully in the 3
rd
phase. This second edition of Neurotransmitter Transporters: Structure,
Function, and Regulation has been put together with the goal of retain-
ing most of the original material in describing for each transporter the
cloning history as well as including new progress in characterizing its
structure, function, regulation, and physiological relevance.
v

vi Preface
Interest in neurotransmitter transporters has increased tremen-
dously, along with clearer understanding of their roles in the patho-
physiology of schizophrenia, Tourette’s syndrome, Parkinson’s
disease, affective disorders, attention deficit/hyperactivity disorder,
neurotoxin accumulation and removal, brain ischemia, amyotropic
lateral sclerosis, or in the mechanism of action of drugs of abuse, anti-
depressants, and antiepileptics. Thus, Neurotransmitter Transporters:
Structure, Function, and Regulation, Second Edition will be of interest to
scientists, graduate students, and advanced undergraduates who seek
a comprehensive overview of this active field in neuroscience. Selected
chapters will also be of interest to physicians who are carrying out
imaging and postmortem measurements of neurotransmitter trans-
porters in the human brain, or physicians who study gene linkages and
polymorphisms in relation to psychiatric and other complex diseases.
Each chapter of Neurotransmitter Transporters: Structure, Function,
and Regulation offers a critical summary and synthesis of the progress
in characterizing each transporter in terms of structure–function rela-
tionships and regulation. Chapters 1–9 focus on various neurotrans-
mitter transporters located in neuronal or glial plasma membranes and
in synaptic vesicles. The Na
+
, Cl
–
-dependent plasma membrane trans-
porters are described for monoamines (Chapters 1–5) and for a number
of compounds including amino acids (GABA, glycine, proline, betaine,
taurine, and creatine (Chapters 6 and 7). The separate family of Na
+
-
dependent glutamate transporters is discussed (Chapters 7 and 8) as
well as the family of vesicular transporters for monoamines, acetylcho-
line, and GABA/glycine (Chapter 9). Chapters 10–13 cover a variety
of issues relevant to transporter structure and function. Chapter 10
describes posttranslational modifications with their important impact
on the function of various transporters. Chapter 11 covers the various
classes of blockers for the dopamine transporter with detailed discus-
sion of structural determinants, and Chapter 12 describes the use of
various in vivo imaging techniques and ligands for biogenic amine
transporters, in particular the dopamine transporter in the human
brain. In Chapter 13, the final chapter, the focus is on dopamine trans-
porter changes in human brain as a result of cocaine administration,
with both in vitro and in vivo imaging approaches.
A number of differences between this second edition and the origi-
nal book can be highlighted. First, the more basic cloning and pharma-
cological information on monoamine transporters, previously in
Chapter 1, can now be found in Chapters 3 and 4. The previous topic
of regulation of serotonin transporters (currently Chapter 1, previ-

Preface vii
ously Chapter 2) has been widened to include all three biogenic amine
transporters, and it now emphasizes phosphorylation and trafficking
phenomena. Three new chapters on biogenic amine transporters have
been added: Chapter 3 on chimera and site-directed mutagenesis stud-
ies, Chapter 4 on gene organization and the relationship of polymor-
phisms with psychiatric and other complex human diseases, and
Chapter 5 on transgenic animals carrying altered genes for plasma
membrane monoamine transporters. Current excitement about chan-
nel properties of transporters is covered in Chapters 2 and 8 regarding
electrophysiological studies on cloned monoamine and amino acid
transporters, respectively. The new chapter on transporter imaging,
Chapter 12, entirely focuses on human results including effects of aging
and brain injury, and changes in schizophrenia, phobia, drug abuse,
and other complex human diseases. Chapter 13 covers changes in
dopamine transporters in human brain as a result of cocaine exposure
as previously, with added discussion of in vivo imaging approaches
for the dopamine transporter included in this update. The previous
final chapter detailing the role of biogenic amine transporters in in
vivo and in vitro monoamine release studies has been omitted as more
recent material on in vivo results is now presented in the new Chapter 5.
The authors of the present chapters have been instrumental in
advancing our knowledge of transporters by their experimental and
conceptual contributions to the field, and I feel fortunate to have been
able to join all their forces together in this second edition. I thank
Paul Dolgert, Tom Lanigan, Sr., Elyse O’Grady, Craig Adams, and
Mark Breaugh at Humana Press for allowing the opportunity of a sec-
ond edition, and I hope the book will continue to be used as a popular
resource in the field.
Maarten E. A. Reith

ix
Contents
Preface.........................................................................................................v
Contributors..............................................................................................xi
1•Regulation of Monoamine Transporters:
Regulated Phosphorylation, Dephosphorylation,
and Trafficking................................................................................ 1
Sammanda Ramamoorthy
2•Mechanisms of Biogenic Amine
Neurotransmitter Transporters...............................................25
Gary Rudnick
3•Structure-Function Relationships
for Biogenic Amine Neurotransmitter Transporters...........53
Nian-Hang Chen and Maarten E. A. Reith
4•Gene Organization and Polymorphisms
of Monoamine Transporters: Relationship
to Psychiatric and Other Complex Diseases............................. 111
Maureen K. Hahn and Randy D. Blakely
5•Monoamine Transporters:
Their Role in Maintaining Neuronal Homeostasis.................. 171
Raul R. Gainetdinov and Marc G. Caron
6•Family of Sodium-Coupled Transporters for GABA,
Glycine, Proline, Betaine, Taurine, and Creatine:
Pharmacology, Physiology, and Regulation.............................. 193
Scott L. Deken, Robert T. Fremeau, Jr.,
and Michael W. Quick
7•Sodium-Coupled GABA and Glutamate Transporters:
Structure and Function.............................................................. 235
Baruch I. Kanner

8•The High-Affinity Glutamate and Neutral Amino-Acid
Transporter Family: Structure, Function,
and Physiological Relevance...................................................... 255
Yoshikatsu Kanai, Davide Trotti, Urs V. Berger,
and Matthias A. Hediger
9•Vesicular Neurotransmitter Transporters:
Pharmacology, Biochemistry, and Molecular Analysis............ 313
Rodrigo Yelin and Shimon Schuldiner
10•Neurotransmitter-Transporter Proteins:
Post-Translational Modifications.............................................. 355
Amrat P. Patel and Maarten E. A. Reith
11•Dopamine-Transporter Uptake Blockers:
Structure-Activity Relationships.............................................. 381
F. Ivy Carroll, Anita H. Lewin, and S. Wayne Mascarella
12•Imaging the Brain Dopamine Transporter
Using PET and SPECT............................................................433
S. John Gatley, Nora D. Volkow, Joanna S. Fowler,
Yu-Shin Ding, Jean Logan, Gene-Jack Wang,
Christoph Felder, Frank W. Telang,
and Andrew N. Gifford
13•In Vitro and In Vivo Imaging
of the Human Dopamine Transporter
in Cocaine Abusers..................................................................467
Deborah C. Mash and Julie K. Staley
Index........................................................................................................503
x Contents

Contributors
URS V. BERGER•Department of Medicine, Brigham and Women's
Hospital, Harvard Institutes of Medicine, Boston, MA
R
ANDY D. BLAKELY•Department of Pharmacology, Center for Molecular
Neurosciences, Vanderbilt University Medical Center, Nashville, TN
M
ARC G. CARON•Department of Cell Biology, Howard Hughes Medical
Institute Laboratories, Duke University Medical Center, Durham, NC
F. I
VY CARROLL•Chemistry and Life Sciences, Research Triangle Institute,
Research Triangle Park, NC
N
IAN-HANG CHEN•Department of Pharmacology, School of Basic
Medical Sciences, Nanjing Medical University, Nanjing,
Jiangsu, China
S
COTT L. DEKEN•Department of Neurobiology, University of Alabama
at Birmingham, Birmingham, AL
Y
U-SHIN DING•Department of Medicine, Brookhaven National
Laboratory, Upton, NY
C
HRISTOPH FELDER• Department of Chemistry, Brookhaven National
Laboratory, Upton, NY
J
OANNA S. FOWLER• Department of Chemistry, Brookhaven National
Laboratory, Upton, NY
R
OBERT T. FREMEAU, JR.•Department of Neurology & Physiology,
University of California at San Francisco School of Medicine,
San Francisco, CA
R
AUL R. GAINETDINOV•Howard Hughes Medical Institute Laboratories,
Department of Cell Biology, Duke University Medical Center,
Durham, NC
S. J
OHN GATLEY• Department of Medicine, Brookhaven National
Laboratory, Upton, NY
A
NDREW N. GIFFORD•Department of Medicine, Brookhaven National
Laboratory, Upton, NY
M
AUREEN K. HAHN• Department of Pharmacology, Center for Molecular
Neurosciences, Vanderbilt University Medical Center, Nashville, TN
M
ATTHIAS A. HEDIGER•Department of Medicine, Brigham and Women's
Hospital, Harvard Institutes of Medicine, Boston, MA
xi

xii Contributors
Y
OSHIKATSU KANAI•Department of Pharmacology, Kyorin University,
Tokyo, Japan
B
ARUCH I. KANNER•Department of Biochemistry, Hadassah Medical
School, The Hebrew University, Jerusalem, Israel
A
NITA H. LEWIN•Chemistry and Life Sciences, Research Triangle
Institute, Research Triangle Park, NC
J
EAN LOGAN•Department of Chemistry, Brookhaven National Laboratory,
Upton, NY
S. W
AYNE MASCARELLA•Chemistry and Life Sciences,
Research Triangle Institute, Research Triangle Park, NC
D
EBORAH C. MASH•Department of Neurology, University of Miami
School of Medicine, Miami, FL
A
MRAT P. PATEL•Division of Treatment and Research Development,
NIDA/NIH, Bethesda, MD
M
ICHAEL W. QUICK•Department of Neurobiology, University of Alabama
at Birmingham, Birmingham, AL
S
AMMANDA RAMAMOORTHY •Department of Physiology/Neuroscience,
Medical University of South Carolina, Charleston, SC
M
AARTEN E. A. REITH•Department of Biomedical and Therapeutic
Sciences, University of Illinois College of Medicine, Peoria, IL
G
ARY RUDNICK•Department of Pharmacology, Yale University School
of Medicine, New Haven, CT
S
HIMON SCHULDINER•Alexander Silberman Institute of Life Sciences,
The Hebrew University, Jerusalem, Israel
J
ULIE K. STALEY•Department of Psychiatry, Yale University,
West Haven, CT
F
RANK W. TELANG• Department of Chemistry, Brookhaven National
Laboratory, Upton, NY
D
AVIDE TROTTI•Department of Medicine, Renal Division, Brigham
and Women's Hospital, Boston, MA
N
ORA D. VOLKOW• Department of Medicine, Brookhaven National
Laboratory, Upton, NY
G
ENE-JACK WANG•Department of Chemistry, Brookhaven National
Laboratory, Upton, NY
R
ODRIGO YELIN• Novel Genomics, Compugen Ltd., Tel-Aviv, Israel

1
Regulation of Monoamine Transporters
Regulated Phosphorylation,
Dephosphorylation, and Trafficking
Sammanda Ramamoorthy
1. INTRODUCTION
The monoamines that include dopamine (DA), norepinephrine (NE), and
serotonin (5-hydroxytryptamine, 5-HT) act as neurotransmitters in peripheral
and central nervous systems (CNS) in a variety of physiological (cognitive,
autonomic, and emotional) functions (1–6). Synaptic neu–rotransmission in
the central nervous system requires the precise control of the duration and the
magnitude of neurotransmitter action at specific molecular targets (7–9). At
the molecular level, neurotransmitter signaling is dynamically regulated by a
diverse set of macromolecules, including biosynthetic enzymes, secretory pro-
teins, ion channels, pre- and postsynaptic receptors and transporters. The cat-
echolamines DA and NE, which are derived from tyrosine, and the indolamine
5-HT, which is derived from tryptophan, are packaged into synaptic vesicles
and released into synapses in response to depolarizing stimuli to activate pre-
and postsynaptic receptors and elicit synaptic responses. Monoamine trans-
porters localized near sites of neurotransmitter release remove transmitters
from the synaptic cleft and its vicinity, and transport them back into presynap-
tic neurons. Monoamine transporters belong to the Na
+
, Cl
–
-dependent,
gamma-amino butyric acid (GABA)/norepinephrine transporter (GAT1/NET)
gene family, which also includes transporters for proline (PROT), taurine
(TauT), glycine (GLYT), creatine (CreaT), betaine (BGT), and other “orphan”
transporters(10–12). Monoamine transporters are the sites of action for widely
used antidepressant drugs and are also high-affinity molecular targets for drugs
of abuse including cocaine, amphetamine, and (+)-3,4-methylenedioxymeth-
amphetamine (MDMA or “ecstasy”) (13-15) (see also Chapter 3). Drugs that
modulate the activity of biogenic monoamine transporters produce profound
behavioral effects, leading to their therapeutic use in the treatment of depres-
1
From:Contemporary Neuroscience:
Neurotransmitter Transporters: Structure, Function, and Regulation, 2nd Edition
Edited by: M. E. A. Reith © Humana Press Inc., Totowa, NJ

2 Ramamoorthy
sion, obsessive-compulsive disorder (OCD), and other mental diseases, and also
to their abuse as stimulants (16–22). Recent molecular and pharmacological
analyses of amine-transporter-knockout mice have confirmed the physiological
importance and essential expression of presynaptic amine transporters for nor-
mal transmitter clearance, presynaptic transmitter homeostasis, and postsynap-
tic/drug responses (23–34) (see also Chapter 5). In particular, functional loss of
DAT through gene knockout (24–29,31–33,35,35) results in profound physi-
cal, physiological, and behavioral changes. Altered transporter function or den-
sity has been implicated in various types of psychopathology, including
depression, suicide, anxiety, aggression, and schizophrenia (17,21,22,37,38).
Although there is no evidence yet for an abnormality in the coding regions of
DAT or serotonin transporter (SERT), a point mutation in the coding region of
human NET has been associated with peripheral Orthostatic Intolerance (OI)
(39) (see also Chapter 4). Two polymorphic regions have been identified in the
SERT promoter that have been implicated in anxiety, mood disorders, alcohol
abuse, and various neuropsychiatric disorders (40–44) .
Reuptake of monoamine neurotransmitters into presynaptic terminals via
transporters is the principal mechanism for terminating monoaminergic neu-
rotransmission. Thus, changes in the activity or expression of the transporters
should have a significant impact on the duration and concentration of monoam-
ines present in and around the synaptic cleft. These changes, in turn, should
influence pre- and postsynaptic responses to released monoamines. This chap-
ter summarizes recent progress in understanding the molecular regulation of
neurotransmitter transporters, with a particular focus on intracellular trafficking
and post-transcriptional regulation by phosphorylation. Changes in monoam-
ine-transporter function or expression reviewed here suggest that cellular pro-
tein kinase(s), protein phosphatase(s), and interacting protein(s) have a potential
role in the regulation of transporters for appropriate transporter function and
expression. Over the past few years, the scientific research in this area has
entered a new age of discovery. Recent studies have revealed that regulation of
transporter function and surface expression are rapidly modulated by “intrinsic”
transporter activity. Furthermore, drugs that block uptake in previously uniden-
tified ways modulate transporter regulation and cell-surface expression.
2. GENERAL MODELS FOR MONOAMINE-TRANSPORTER
REGULATION
Neural activity, hormones, growth factors, environmental factors, and
pharmacological agents regulate monoamine uptake, specific radioligand
binding, and mRNA levels (discussed for SERT in Chapter 2, 1st edition
[45]). These studies suggest the presence of endogenous regulatory mecha-
nisms that influence transporter expression and function at the level of gene

Regulation of Monoamine Transporters 3
transcription and translation (long-term or chronic regulation requires hours
or days) or post-translational protein modifications (short-term or acute
regulation requires seconds to minutes) (Fig. 1).
2.1. Long-Term Regulation of Monoamine Transporters
at the Gene Level
The structural analysis of transporter-gene-promoter regions reveals a
number of canonical transcription binding sites that may be important in
controlling the responses of the transporter genes to regulatory factors (46–
51). Notably, binding sites for transcription factors including TATA-like
m
otif, an AP1 site, element for CREB binding (CRE), AP-2, NF-IL6, NF-
Fig. 1. Life cycle of the monoamine amine transporter. Neurotransmitter clear-
ance is a highly orchestrated process involving regulation of transporter-gene
expression and post-translational modifications. Promoter of transporter gene con-
tains several canonical transcription binding sites for constitutive and regulated
transcription factors, which drive transcription of transporter in the nucleus. Fol-
lowing transcription, transporters are translated and delivered to their specific mem-
brane sites of expression in cells. At the surface level, the retention and functional
expression of transporter proteins are regulated by cellular protein kinases and phos-
phatases. Depending upon the signals, the transporter may enter into the recycling
pool, allowing the transporter to return to the cell surface or enter into lysosomal
degradative pathways.

4 Ramamoorthy
Kβ, and SP1 sites have been identified in the SERT-promoter region
(46,47,50,51). Relevant investigations performed on JAR cells have shown
that activation of cAMP-dependent (mimicked by cholera toxin, forskolin,
dibutryl cAMP) and independent pathways (mimicked by staurosporine,
interleukin-1β) increases 5-HT uptake. Increases in 5-HT uptake require pro-
longed stimulation (4-h) and messenger RNA and protein synthesis (52–54).
Since the long-term modulation of SERT expression both in vivo and in
vitro is described in detail in an earlier chapter (45), only recent develop-
ments in the long-term regulation of SERT are summarized here. Unlike
SERT, very little is known about NET and DAT gene regulation. Earlier in
vivo and in vitro studies demonstrate modulation of NE uptake by several
factors such as insulin, atrial natriuretic peptide (ANP), angiotensin (ANG
II), dexamethasone, nerve-growth factor (NGF), and pharmacological sub-
stances such as desipramine and cocaine (55–61). Altered NET mRNA lev-
els are shown for the NET modulation by insulin, dexamethasone, NGF,
desipramine, and cocaine. Recent reports show upregulation of DAT and NET
genes by cocaine treatment. During pregnancy, cocaine exposure results in
increased DAT mRNA levels in the fetal rhesus monkey brain (62). Another
study revealed increased levels of NET mRNA in the placentas of rats treated
with cocaine at the mRNA level (63,64). Although the actual cellular signal-
ing pathways responsible for such regulation at the genetic level have not yet
been identified, these studies suggest a significant functional role for the
monoamine transporters in fetal development.
2.2. Acute Regulation of Monoamine Transporters
Amino-acid sequence analysis of SERT, DAT, and NET proteins reveals
numerous consensus sites for protein kinases as well as putative interactive
motifs in intracytoplasmic domains, suggesting that second messengers play
a role in the post-translational regulation of monoamine transporters (65–
70). Prior to the cloning of monoamine transporters, many studies suggested
a pivotal role for second messenger-linked pathways in acute modulation of
neurotransmitter uptake (45,71) . Potential pathways through which second
messenger-linked signals might regulate transporter function acutely are il-
lustrated in Figs. 1 and 2. Regulation of transporters occurs directly by phos-
phorylation of transporter proteins. Evidence indicates that phosphorylation
of transporter proteins changes intrinsic transporter activity, alters trans-
porter turnover, regulates fusion of transporter containing vesicles with the
plasma membrane, and regulates sequestration of transporter from the
plasma membrane by modulating endocytic pathways. Alternatively, regu-
lation of transporters could occur through their interaction with other pro-
teins by phosphorylation-dependent/or -independent pathways (72).

Regulation of Monoamine Transporters 5
2.3. Regulation of Monoamine-Transporter Surface Expression
A common approach has been employed in studying acute transporter
regulation in endogenous and heterologous systems by treatment with vari-
ous activators and inhibitors of protein kinases and phosphatases. The most
consistent finding is that activators of protein kinase C (PKC) or the agents that
maintain the phosphorylation state, such as phosphatase inhibitors, rapidly reduce
amine transport capacity. The major kinetic alterations typically observed in acute
modulation paradigms are changes in transport capacity (V
max) with little or no
Fig. 2. Acute kinase-mediated regulation of monoamine-transporter expression.
Monoamine transporters are regulated by multiple signaling pathways linked to
receptor-triggered kinase activation. Transporter phosphorylation may be compart-
mentalized in a manner, appropriate for its use as a signal for transporter traffick-
ing. Phosphorylation and dephosphorylation cascades regulate transporter
expression. Direct phosphorylation may change transporter function by multiple
discrete mechanisms. For example, phosphorylation of the transporter may A)
change its intrinsic transport activity, B) regulate sequestration of transport from
plasma membrane by modulating endocytosis machinery, C) regulate exocytic
fusion of transporter-containing vesicles with the plasma membrane. Transporter-
associated proteins such as protein phosphatase 2A would govern the stoichiom-
etry of amine-transporter phosphorylation as well as its duration, providing an
important trigger for transporter expression and trafficking.

6 Ramamoorthy
significant change in substrate affinity (K
m). In a variety of preparations, in-
cluding synaptosomes and cell lines, application of the PKC activator, β-
PMA, selectively reduces the V
max for transport of DA, NE, and 5-HT (73–80).
Several control experiments confirm the specificity of β-PMA on PKC acti-
vation and, thus, the modulation of monoamine transporters by PKC is not a
generalized effect. First, β-PMA has no, little, or opposite effects on other
transporter activities (78,81). Second, the effects of β-PMA are stereospe-
cific; the active stereoisomer of PMA, but not the inactive alpha isomer,
modulates monoamine-transporter function (78). Third, the β-PMA effect can
be blocked by PKC inhibitors, such as staurosporine and bisindolylmalemide
(78). Finally, the β-PMA effect is paralleled by an activation and transloca-
tion of PKC from the cytoplasm to the plasma membrane (82,83).
The reduction in transport capacity (V
max) suggests silencing of plasma-
membrane resident amine transport protein or decreased cell-surface expres-
sion. Initial evidence for altered surface expression of transporter protein by
PKC has come from homologous GAT1 GABA transporters expressed in
Xenopus oocytes. In Xenopus oocytes, phorbol esters increase GAT1 activity
by translocating GAT1 from intracellular compartments to the cell surface
(84,85). The first evidence for altered cell-surface expression of a monoam-
ine transporter as the result of PKC activation has come from studies of hu-
man SERT stably expressed in Human Embryonic Kidney (HEK-293) cells
(78). In this study, immunoprecipitation following cell-surface protein
biotinylation was employed to quantify the cell-surface SERT protein pool
before and after PKC activation. Exposure to β-PMA leads to 30–40% loss
of SERT proteins on the plasma membrane in parallel with reductions in 5-
HT uptake capacity. A concomitant increase in the intracellular SERT pool
suggests translocation of SERT from the plasma membrane to the intracellu-
lar pool. Similar to 5-HT uptake, loss of cell-surface SERT was blocked by
the PKC inhibitor staurosporine (78). The protein phosphatase inhibitor,
okadaic acid, also regulates SERT activity, suggesting that cellular phos-
phatases monitor phosphorylation-dependent SERT regulation in this cell
model(86,87). Similar observations were made in LLC-PK1 cells stably
transfected with human NET (73,74), DAT expressing PC12, African Green
Monkey kidney (COS), and Madin-Darby canine kidney (MDCK) cells
(79,88–90), and DAT cRNA-injected Xenopus oocytes following PKC acti-
vation(80). Using PC12 cells stably transfected with human DAT, Melikian
and Buckley (89) reported that activation of PKC results in decreased uptake
capacity in concert with decreased cell-surface DAT density. The study also
showed that internalized DAT is colocalized with transferrin receptors, which
are known to actively recycle to the plasma membrane, although no supporting
data were provided. The authors suggested that the PKC-dependent, internal-

Regulation of Monoamine Transporters 7
ized DAT may recycle to the plasma membrane. In another study, using green
fluorescent protein (GFP)-tagged DAT stably expressed in MDCK cells,
Daniels and Amara (88) elegantly visualized rapid DAT internalization in live
cells following PKC activation. PKC-dependent DAT internalization is not a
general increase in vesicular traffic from the cell surface, because another cell-
surface protein–E-cadherin–did not internalize under similar conditions. Inter-
estingly, a dominant-negative mutant of dynamin 1, which has been shown to
block clathrin-mediated endocytosis, is able to block PKC-dependent DAT
internalization. These observations suggest that PKC regulates DAT internal-
ization by clathrin-mediated and dynamin-dependent cellular mechanisms.
They also provide evidence that internalized DAT is targeted to endosomal/
lysomal pathways for degradation. The molecular mechanisms responsible for
various PKC-dependent DAT internalization pathways (recycling endosomes
vs degradative lysosomes) in these studies are unknown. Another signaling
pathway mediated by cAMP-dependent protein kinase A (PKA) upregulates
striatal DAT by increasing V
max(91). Pristupa and colleagues (77) showed a
bidirectional trafficking of DAT by PKC and PKA pathways in DAT-trans-
fected COS and Sf9 cells (77, 91–93). The studies described above used heter-
ologous expression systems transfected with amine transporter cDNAs or
Xenopus oocytes injected with amine transporter cRNA. Studies with isolated
preparations such as synaptosomes, brain slices, or platelets also reveal similar
changes in kinetic parameters following acute regulation of amine transporter
by PKC and other kinases (90,91,94–99). Relatively few studies have demon-
strated amine-transporter regulation by presynaptic receptors (25,100–103).
These observations establish that rapid shuttling of amine transporters between
plasma membrane and intracellular compartments, or distribution of transport-
ers in active and inactive pools, provide a route through which amine uptake
may be regulated by second messenger-linked systems, possibly following ac-
tivation of presynaptic auto/ or hetero receptors (Figs. 1 and 2).
3. TRANSPORTER PHOSPHORYLATION: A SIGNAL
FOR AMINE UPTAKE MODULATION
Recent observations have suggested that modulation of amine transport-
ers followed by activation of cellular protein kinases or inhibition of protein
phosphatases leads to amine transporter phosphorylation. These observations
support the role of amine-transporter phosphorylation in transporter regula-
tion. Recently, Ramamoorthy et al. (8) demonstrated that SERT proteins are
phosphorylated under basal conditions in HEK-293 cells, and that phosphory-
lation is increased in a time- and dose-dependent manner by activators of PKC,
and PKA, as well as inhibitors of phosphatase 1/2A. β-PMA-triggered SERT

8 Ramamoorthy
phosphorylation is blocked by the PKC inhibitors staurosporine and
bisindolylmalemide, but not by PKA inhibitors. However, SERT phosphory-
lation triggered by cyclic AMP analogs or cholera toxin can be blocked by
PKA inhibitors but not by PKC inhibitors. Protein phosphatase (PP1/PP2A)
inhibitors such as okadaic acid and calyculin A, but not the calcineurin (PP2B)
inhibitor cyclosporin, significantly elevate SERT phosphorylation to a similar
extent as observed with β-PMA. The elevation of SERT phosphorylation
induced by β-PMA and okadaic acid is positively correlated with losses in 5-
HT transport, suggesting an extremely close correlation between SERT phos-
phorylation and 5-HT uptake capacity. Additivity of PKC-triggered SERT
phosphorylation is found after cotreatment with okadaic acid, β-PMA, and
cholera toxin at maximally effective concentrations, suggesting that distinct
sites are being phosphorylated as a result of the activation of PKC, PKA, and
as yet unidentified endogenous kinase(s). Interestingly, the antagonists for
PKC, PKA, CaM kinase II, and depletion of intracellular Ca
2+
fail to reduce
SERT phosphorylation observed in the presence of the PP1/PP2A inhibitor.
These data suggest that protein kinase(s) other than PKC, PKA, PKG, or Ca
2+
-
dependent kinase—yet to be identified—phosphorylates SERT under basal
conditions in this heterologous model (99). Similar evidence for DAT phos-
phorylation comes from studies using native rat-brain striatal synaptosomes
as well as heterologously expressed rat DAT in COS and LLC-PK1 cells
(90,92,104,105). Similar to human SERT, DAT exhibits basal phosphoryla-
tion that can be augmented by PKC activation and phosphatase inhibition. In
both preparations, PKC activation leads to decreased DAT activity, which is
manifested primarily as a reduction in V
max. PKC-dependent DAT phosphory-
lation was sensitive to the PKC inhibitors staurosporine and
bisindolylmalemide. One important difference between DAT and SERT phos-
phorylation is that okadaic acid-triggered SERT phosphorylation is not
blocked by staurosporine, whereas staurosporine blocks okadaic acid-induced
DAT phosphorylation. Another difference between SERT and DAT phospho-
rylation studies is the evidence that SERT undergoes rapid phosphorylation
following PKA activation, whereas DAT is not phosphorylated by PKA acti-
vation(90,92). These studies suggest that specific kinase-mediated pathways
may influence amine-transporter phosphorylation and regulation in a specific
manner, and may not be shared with each other.
3.1. Intrinsic Transporter Activity Governs Amine-Transporter
Surface Expression
Another important recent finding is the ability of amine-transporter sub-
strates and antagonists to influence transporter trafficking and plasma-mem-
brane residency (86). A common theme is emerging among transporters such

Regulation of Monoamine Transporters 9
as DAT, GAT1, and GLAST, which are regulated by their substrates (106–
109). For example, Ramamoorthy and Blakely (86) provided evidence that
SERT substrates such as 5-HT, amphetamines, fenfluramine, and antago-
nists such as antidepressants and cocaine control PKC-dependent SERT
phosphorylation and surface redistribution (86). This substrate-transporter
feedback loop provides a mechanism by which changes in extracellular neu-
rotransmitter concentrations rapidly modulate neurotransmitter transport
capacity. This study showed that extracellular 5-HT significantly blunts the
SERT phosphorylation achieved by PKC activation. This inhibitory effect
of 5-HT occurs in the presence of 5-HT receptor antagonists, suggesting
that the effect of 5-HT on PKC-mediated SERT phosphorylation is not de-
pendent on 5-HT-receptor activation. Moreover, the 5-HT effect displays a
concentration-dependence similar to that of 5-HT transport. Like 5-HT trans-
port, the 5-HT effect on PKC-induced SERT phosphorylation requires ex-
tracellular Na
+
and Cl
–
, and can be blocked by SSRIs but not by DAT- and
NET-selective antagonists. Diminished SERT phosphorylation cannot be
achieved by preloading the cells with 5-HT prior to PKC activation. Other
SERT substrates such as D-amphetamine and fenfluramine, but not dopam-
ine and norepinephrine, have a similar inhibitory effect on PKC-mediated
SERT phosphorylation. Cell-surface protein biotinylation experiments re-
veal that PKC activation results in decreased cell-surface SERT density, and
this effect can be abolished by the presence of 5-HT during PKC activation
(86). Together, these findings suggest that SERTs in the presence of ac-
tively transporting substrates reduce the opportunity for PKC-linked SERT
phosphorylation and transporter internalization. Thus, control of SERT cell-
surface expression by 5-HT would provide a novel homeostatic mechanism
that may serve in the neuron to fine-tune transport capacity to match de-
mands imposed by fluctuating levels of serotonin. Signaling pathways linked
to presynaptic auto- and hetero-receptors could provide positive/negative
feedback control, and could provide a mechanism by which changes in syn-
aptically released extracellular 5-HT could rapidly modulate SERT capac-
ity. Interestingly, amphetamines can substitute for 5-HT in suppressing
PKC-mediated SERT phosphorylation. Such action could override homeo-
static transporter sequestration processes and provide for psychostimulant-
induced sensitization by increasing the number of psychostimulant targets
available to a subsequent stimulus. On the other hand, nonpermeant SERT
ligands such as selective serotonin reuptake inhibitors (SSRIs) and cocaine,
which prevent 5-HT permeation, block the effect of 5-HT. Thus, SSRIs may
have therapeutic utility in disease states, not only by preventing 5-HT up-
take, but also by shifting the cellular distribution of SERT.

10 Ramamoorthy
3.2. Protein Phosphatase 2A: An Intimate Amine Transporter
Partner in Regulating the State of Transporter Phosphorylation
As described earlier, agents that maintain the phosphorylated state modu-
late amine transporter activity. For example, the protein phosphatase 1/2A
(PP1/PP2A) inhibitor, known as okadaic acid downregulates SERT, DAT
and NET activity (87,90,92) . Treatment with okadaic acid or another potent
PP1/PP2A inhibitor, calyculin A, results in a rapid increase in SERT phos-
phorylation in parallel with a decrease in 5-HT uptake (87). Similarly, these
inhibitors also promote DAT phosphorylation and functional
downregulation in striatal synaptosome preparations (90). Maintenance of
the phosphorylation state and regulation of surface amine-transporter
expression is a balance between the actions and localization of protein
kinases and protein phosphatases. The pronounced effect of PP1/PP2A inhi-
bition on amine uptake and amine-transporter phosphorylation suggests the
possible physical association of PP1/PP2A as a regulatory complex with
amine-transporter proteins. Desensitization and trafficking of several G-pro-
tein-coupled receptors are regulated by phosphorylation-dependent path-
ways. This involves regulated physical association of receptors with kinases
and phosphatases and other adaptor proteins (110–115). For example, the
catalytic subunit of PP2A is believed to play an important role in recycling
β2-adrenergic receptors by associating with phosphorylated and internal-
izedβ2 receptors. The associated PP2Ac catalyzes the dephosphorylation of
β2 receptors, facilitating their recycling to the plasma membrane (113,114).
Recently, Bauman et al. (116) documented the existence of okadaic acid-
sensitive phosphatase activity in the immunoisolated SERTs from stably
transfected HEK-293 cells. Western blots of SERT immunoprecipitates us-
ing monoclonal PP2Ac antibody revealed the presence of PP2A catalytic
subunit. Blots of SERT immunoprecipitations from multiple brain regions
also showed the presence of PP2Ac with SERT as a complex, suggesting
that SERT/PP2Ac exists as an associated complex in vivo. Qualitatively,
similar transporter and PP2Ac associations were found for DAT and NET
proteins. Evaluation of DAT and NET immunoprecipitates from rat striatal
synaptosomes and vas deferens, respectively, shows the presence of PP2Ac
as a complex with DAT and NET proteins (12). Together, these findings
suggest that PP2Ac associations with biogenic amine transporters are also
likely to occur with other transporters of the gene family, and this associa-
tion plays an important role in their regulation. Another hypothesis is that
the associated PP2Ac activity may govern the stoichiometry of amine-trans-
porter phosphorylation, as well as the duration of the phospho-nature of
transporter. This will provide an important trigger for transporter-protein

Regulation of Monoamine Transporters 11
expression and trafficking. In addition, the association of PP2Ac with amine
transporters may be dynamically regulated by kinases/phosphatases and by
the state of transporter phosphorylation. In support of this hypothesis, trans-
porter and PP2Ac associations can be regulated by PKC activation, phos-
phatase inhibition, and transporter substrates. In 293-hSERT cells, treatment
of cells with β -PMA decreases SERT-associated PP2Ac, which is blocked
by the PKC inhibitors staurosporine, and bisindolylmalemide. Under simi-
lar conditions, β-PMA treatment leads to increased SERT phosphorylation
and internalization (86,87). These results suggest that PKC-dependent SERT
phosphorylation and internalization occurs by dissociating PP2Ac from a
pre-existing PP2Ac/SERT complex. Dissociation of PP2Ac from SERT
leads to decreased SERT-dephosphorylation, contributing to enhanced
SERT phosphorylation. 5-HT blocks the PKC-triggered dissociation of
PP2Ac from SERT, suggesting that transport or the transporter provide a
signal for SERT phosphorylation and association of other proteins. How-
ever, it is presently not known whether PKC-dependent SERT phosphoryla-
tion occurs first—which in turn triggers PP2Ac dissociation—or whether
PKC-dependent SERT phosphorylation is dependent on PP2Ac dissocia-
tion. In addition to PKC-dependent-regulated association of the SERT/
PP2Ac complex, the PP1/PP2A inhibitors, okadaic acid or calyculin A, dis-
assemble the PP2Ac/SERT complex. This regulation is specific and depen-
dent on PP1/PP2A inhibition, but not by inhibiting other phosphatases. Other
phosphatase inhibitors—such as cyclosporine A, which is specific for
PP2B—have no ability to regulate the PP2Ac/SERT complex. Analogous to
SERT studies, PP2Ac association with NET in vas deferens is also decreased
by PKC activation and PP1/PP2A inhibition (116). These results suggest
several possibilities in the mode of SERT association with PP2Ac. (1)
Okadaic acid is known to bind the active site of PP2Ac and, thus, okadaic
acid may compete with SERT for the same site on PP2Ac. This also sug-
gests that the PP2Ac active site may be the motif by which PP2Ac assembles
with SERT directly or via other adaptor protein(s). (2) Protecting phospho-
SERT by inhibiting dephosphorylation of SERT by okadaic acid could trig-
ger release of PP2Ac from the SERT complex. (3) Okadaic acid-linked
inhibition of phosphatase leads to activation of kinase, which could trigger
the dissociation of PP2Ac from the SERT complex.
3.3. Regulation of Biogenic Amine Transporters by Protein Interactions
Although the direct phosphorylation and dephosphorylation of transport-
ers by cellular protein kinases/phosphatases is involved in the dynamic regu-
lation and expression of transporters, other integral membrane proteins also
appear to play an important role in trafficking and the catalytic function of

12 Ramamoorthy
transporters. The first evidence for the physical association of GAT1 GABA
transporters with the (t)-SNARE protein, syntaxin-1A, has come from stud-
ies by Quick and colleagues using native and heterologous expression model
systems(86,117). Interestingly, this association is modulated in a PKC-
dependent manner, and the interaction of GAT1 with Syntaxin-1 is required
for GAT1 regulation by PKC activation. Syntaxin-1A association not only
regulates PKC-dependent trafficking of GAT1, but also appears to influ-
ence the catalytic function of the transporter (85,117). Recently, it has been
shown that syntaxin-1A interacts with the N-terminal cytoplasmic domain
of the GAT1 GABA transporter and decreases transport rates (118). Co-
immunoprecipitation of syntaxin-1A with the glycine transporter, GLYT2,
has been shown in heterologous and native systems (119) . Like GAT1 and
GLYT2, NET appears to be physically associated with syntaxin-1A. Using
noradrenergic tissues such as vas deferens and transfected cell lines, Sung et
al.(120)reported that syntaxin-1A is found in NET immunoprecipitates.
Activation of PKC or phosphatase inhibition decreases the syntaxin-1A level
in the NET immunoprecipitates. Altering intracellular Ca
2
+ also regulates
the syntaxin/NET interaction. Together, these findings suggest an important
role for syntaxin-1A in neurotransmission by regulating both transmitter
release and reuptake. In addition, signals following presynaptic receptor
stimulation may influence transporter function and expression by regulating
the stability of transporter heteromeric complexes. Recently, Torrres et al.
(121)showed the interaction of the PDZ domain-containing protein, PICK1
(protein interacting with C kinase) with C-terminal cytoplasmic domains of
DAT and NET (121). Co-expression of DAT and PICK1 in HEK-293 cells
enhances DAT activity and PICK1 co-immunoprecipitates with DAT. Fur-
thermore, confocal microscopy analysis revealed colocalization of both
DAT and PICK1 in a heterologous system and in dissociated dopaminergic
neurons(104). Since PICK1 is a PKC α-binding protein, it is possible that
PICK1 acts like an adaptor to bring PKC near DAT proteins, which in turn
could regulate PKC-evoked DAT phosphorylation, trafficking, and func-
tion. Lee and colleagues (122) recently identified the interaction of the C-
terminal cytoplasmic domain of DAT with α-synuclein.α-Synuclein is
enriched in dopaminergic nerve terminals and has been implicated in
Parkinson’s and other neurodegenerative disorders. α-Synuclein binds
directly to the C-terminal region of DAT and facilitates the plasma-mem-
brane clustering and increase in DAT activity. This study suggests that the
α-synuclein/DAT complex regulates normal dopaminergic neurotransmis-
sion, and altered interactions may result in abnormal DAT function, causing
the dopaminergic neurodegeneration seen in Parkinson’s disease (122).

Regulation of Monoamine Transporters 13
In summary, evidence suggests that amine-transporter levels may be
modulated by hormones, growth factors, neuronal activity, and pharmaco-
logical agents. The intracellular second messenger systems mimic cellular
protein kinases and phosphatases, and subsequently regulate amine-trans-
porter gene and protein expression. The past few years represent an active
period in transporter research related to structure, expression, function, and
regulation of amine transporters. These investigations undoubtedly provide
strong support for the idea that amine transporters are principal players in
regulating normal and abnormal amine signaling in the CNS and periphery,
and, thus, complex behavioral and physiological functions. However, even
more exciting is the discovery that the intrinsic transport capacity of a trans-
porter molecule governs its own plasma-membrane expression and function
(86,106,107). There appear to be multiple mechanisms by which transporter
substrates and antagonists can influence transporter function and expres-
sion. The capacity of transporters to fine-tune their function in response to
extracellular neurotransmitter levels would result in maintaining a constant
level of neurotransmitter at the synaptic cleft. In other words, increased cell-
surface expression of neurotransmitter transporter following an increase in
the extracellular level of neurotransmitter would, in turn, promote the rapid
clearance of released neurotransmitter from the synaptic cleft and provide a
mechanism to maintain the synaptic concentrations of neurotransmitter level
within a narrow range (Fig. 3). It is interesting to know how the architecture
of trafficking mechanisms is designed in nature at pre- and postsynaptic
levels for the termination of neurotransmission (Fig. 3). At the postsynaptic
level, receptors for neurotransmitters internalize following agonist-induced
phosphorylation. The cascade of events lead to receptor desensitization and
termination of neurotransmission. At the presynaptic level, the active termi-
nation of neurotransmission is mediated by plasma-membrane transporters.
In contrast to what happens at the postsynaptic level (decrease in receptor
density caused by internalization following agonist-induced phosphoryla-
tion), increased transporter density at the presynaptic membrane in response
to synaptic neurotransmitter appears to be another mechanism for rapid ter-
mination of neurotransmission. The coordinate regulatory pattern of trans-
porter and receptor trafficking at pre- and postsynaptic levels may provide
normal neurotransmission. An altered pattern or a disturbance in the pre-
and postsynaptic regulatory mechanisms could lead to abnormal neurotrans-
mission, and thus abnormal behavior or brain disorders. Drugs of abuse such
as cocaine and amphetamines bind and inhibit biogenic amine transporters,
and may interfere with activity-dependent regulatory mechanisms, and may
cause or initiate drug addiction or sensitization. Along with information
delineatingcis/trans acting elements and signals for acute and chronic

14 Ramamoorthy
Fig.3. Hypothetical model for the phosphorylation-dependent coordinate trafficking of presynaptic monoamine transporters and
postsynaptic receptors in normal neurotransmission. Phosphorylated presynaptic monoamine transporter —for example, SERT—enters
into a regulated endocytosis pathway. Internalized SERT could be dephosphorylated by associated protein phosphatase, allowing t he
return of nonphosphorylated SERT to the cell surface. SERT substrate 5-HT increases SERT plasma-membrane residency time by not
allowing the SERT to be phosphorylated and internalized. Increased cell-surface retention of the transporters may regulate syna ptic
transmission by efficient clearance of released neurotransmitters. This may serve as a built-in “feed-back” mechanism to autoregulate
the transporters and suppress the influence of presynaptic receptor-linked stimuli. In contrast to increase in presynaptic tran sporter
following neural activity, postsynaptic receptors are internalized following agonist induced phosphorylation, resulting in rece ptor desen-
sitization. Thus, the regulation of trafficking and expression of transporters at the presynaptic level and of receptors at the postsynaptic
level may be executed in a coordinate way, providing normal neurotransmission.

Regulation of Monoamine Transporters 15
amine-transporter regulation, over the next few years with hard work and
ingenuity, we will expect rapid progress in understanding the role of cellular
regulation of amine transporters in amine signaling and behavior. These
developments provide opportunities to add further impetus to the drive for
psychotherapeutic innovation.
ACKNOWLEDGMENTS
Support from the National Alliance of Research on Schizophrenia and
Depression (NARSAD) and the developmental fund from the Medical Uni-
versity of South Carolina are greatly acknowledged. Thanks to Dr. J. F.
McGinty for critical review of this chapter. Thanks also go to Dr. Reith for
providing this wonderful opportunity to contribute this chapter, and for his
encouragement.
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Regulation of Monoamine Transporters 23
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25
2
Mechanisms of Biogenic Amine
Neurotransmitter Transporters
Gary Rudnick
1. INTRODUCTION
The biogenic amine transporters, as described in Chapters 1, 3, and 5 of
this book, terminate the action of released biogenic amine neurotransmit-
ters. These transporters utilize norepinephrine (NE), dopamine (DA), and
serotonin (5-HT), and are referred to as NET, DAT, and SERT, respectively.
Interruption of their function by agents such as antidepressants and stimu-
lants causes profound changes in mood and behavior. In addition to their
importance in regulating the extracellular concentration of neurotransmit-
ters, these proteins are fascinating molecular machines that utilize the energy
from transmembrane ion gradients to accumulate intracellular neurotrans-
mitters. The pharmacology and molecular biology of these proteins is well
covered in Chapters1, 3, 5, and 11. This chapter focuses on the mechanism
of neurotransmitter transport. We are still a long way from completely
understanding how these proteins work. However, recent advances have
given us further insight into this process, and encourage the hope that cur-
rent and future research will provide a more complete understanding of the
transport mechanism.
2. NA, K, AND CL IONS: COFACTORS FOR TRANSPORT
Like many mammalian plasma-membrane transport systems, the biogenic
amine transporters require the presence of external Na
+
ions. This phenom-
enon was observed first for epithelial transporters, such as those for glucose
and amino acids (1). The transport of NE into peripheral nerve endings was
also found to be Na
+
-dependent(2), as was 5-HT transport into platelets (3).
When synaptosomes were established as an experimental system for study-
ing presynaptic mechanisms, the Na
+
dependence of neurotransmitter uptake
From:Contemporary Neuroscience:
Neurotransmitter Transporters: Structure, Function, and Regulation, 2nd Edition
Edited by: M. E. A. Reith © Humana Press Inc., Totowa, NJ

26 Rudnick
became firmly established, as each system in turn demonstrated its depen-
dence on extracellular Na
+
(4–9). In all these cases, replacement of Na
+
with
any other ion results in a loss of transport activity.
The importance of this Na
+
requirement became more apparent as the
energetics of transport were studied. The Na
+
requirement provided a way
to understand how the energy of ATP hydrolysis drives transport. ATP is
utilized by the Na
+
pump to move Na
+
ions out of—and K
+
into—the cell.
By incorporating Na
+
into the transport reaction, the neurotransmitter trans-
porters couple the energy released by ATP hydrolysis to the downhill Na
+
flux that accompanies transmitter accumulation (Fig. 1). Because K
+
is
pumped by the ATPase, it is not surprising that some neurotransmitter trans-
porters also utilize internal K
+
in the transport process. The most notable are
the 5-HT and glutamate transporters (10,11), although other systems may
also take advantage of K
+
concentrated inside cells by the Na
+
pump. The
requirement for internal K
+
can be fulfilled by H
+
in the case of the 5-HT
transporter(12), but efforts to demonstrate this phenomenon with glutamate
transporters have resulted in the opposite conclusion—that H
+
equivalents
were moving with the flow of substrate rather than against it (13).
Another consequence of Na
+
pump action is a result of the fact that three
Na
+
ions are pumped out for each two K
+
ions pumped into the cell, leading
to the generation of a transmembrane electrical potential. In itself, this
potential can be used by neurotransmitter transporters, but more specifically,
the potential leads to the loss of Cl
–
ions from the cell. Many neurotransmit-
ter transporters utilize this asymmetric Cl
–
distribution as a driving force.
With the exception of glutamate transporters, all of the neurotransmitter
transporters and many related transporters require Cl
–
as well as Na
+
(14).
For this reason, the neurotransmitter transporter gene family is often referred
to as the NaCl-coupled transporter family (15) . The glutamate transporters
represent a separate gene family.
2.1. Transport and Binding Requirements
One advantage to the study of biogenic amine transporters, relative to
other members of the family, is that high-affinity ligands are available. Tri-
cyclic antidepressants such as imipramine and desipramine bind to SERT
and NET, and have been used to investigate the ion dependence of the bind-
ing process. SERT requires both Na
+
and Cl
–
ions for maximal
[
3
H]imipramine binding (16), although repeated attempts to demonstrate any
effect of K
+
have been negative. Similar results have been obtained with
[
3
H]desipramine and NET (17). Antidepressant binding differs from trans-

Biogenic Amine Transporters 27
port, however. The Na
+
dependence of [
3
H]imipramine binding to SERT
and imipramine inhibition of transport were both sigmoidal, suggesting that
two or more Na
+
ions participate in the reaction (18). In contrast, the Na
+
dependence of 5-HT transport and 5-HT inhibition of [
3
H]imipramine bind-
Fig. 1. Neurotransmitter recycling at the nerve terminal. Neurotransmitter (NT)
is released from the nerve terminal by fusion of synaptic vesicles with the plasma
membrane. After release, the transmitter is transported across the plasma mem-
brane by a Na
+
-dependent transporter in the plasma membrane. Transmitter deliv-
ered into the cytoplasm is further sequestered in synaptic vesicles by a vesicular
transporter using the transmembrane H
+
gradient as a driving force. This driving
force, shown by the arrow pointing in the direction of downhill H
+
movement, is
generated by an ATP-dependent H
+
pump in the vesicle membrane. The Na
+
and
K
+
gradients across the plasma membrane are generated by the Na
+
/K
+
-ATPase.
This enzyme also creates a transmembrane electrical potential (negative inside)
that causes Cl
-
to redistribute. Neurotransmitter transport across the plasma mem-
brane is coupled to the Na
+
, Cl
–
, and K
+
gradients and the membrane potential
generated by the ATPase.

28 Rudnick
ing showed simple saturation behavior consistent with the involvement of
only one Na
+
ion in 5-HT binding and transport (18). Similar results were
obtained with NET. Transport is a simple saturable function of Na
+
(19), but
[
3
H]desipramine binding shows a sigmoidal Na
+
dependence (17).
When binding of other ligands was examined, the difference between sub-
strates and inhibitors became even more obvious. Paroxetine and the cocaine
analogs CFT and β-CIT bind to SERT and inhibit transport. This binding
process was stimulated by Na
+
but not by Cl
–
(20–22), although Cl
–
is
required for transport. The DA transporter also binds cocaine and its ana-
logs(23,24), and has demonstrated a different ion dependence for transport
andβ-CIT binding (24). For both SERT and DAT, β-CIT binding was
inhibited when Na
+
was replaced with Li
+
but did not require Cl
–
, and for
both transporters, β-CIT binding was inhibited at low pH (24). In contrast,
the binding of 5-HT or DA, was stimulated by Cl
–
and not affected by low
pH(24), although neutralization of DA at high pH decreases its binding to
DAT(25). The presence of Li
+
ions apparently favors a conformation of
SERT with a lower affinity for cocaine and its analogs, but with similar 5-
HT affinity (25a). These studies and others clearly indicate that inhibitor
binding may be smilar, in some aspects, to substrate binding. However, the
two processes are distinct in their ionic dependence. Despite the differences,
interactions between inhibitors and substrates at 5-HT, NE, and DA trans-
porters are competitive, at least in equilibrium-binding studies. Thus, a
single binding site, or a set of overlapping binding sites, could account for
substrate and inhibitor binding.
The sensitivity of β-CIT binding to displacement by substrate has allowed
measurements of substrate binding under conditions which preclude direct
substrate-binding measurements. Using [
125
I]β-CIT, 5-HT binding to SERT
was measured in the absence of Na
+
and Cl
–
, and the individual effects of
these ions was determined (22). Although Cl
–
stimulated 5-HT binding by
itself, Na
+
alone actually decreased 5-HT-binding affinity. Maximal 5-HT
affinity was observed only in the presence of both Na
+
and Cl
–
, suggesting
that 5-HT binds to the transporter together with these two ions (22) .
3. ION GRADIENTS DRIVE BIOGENIC AMINES ACROSS THE
MEMBRANE
3.1. Influence of Ion Gradients on 5-HT Transport
Studies with synaptosomes and platelets have indicated that the biogenic
amine transport systems possess an impressive ability to concentrate DA,
NE, and 5-HT. However, these preparations contained intracellular amine

Biogenic Amine Transporters 29
storage organelles (synaptic vesicles or dense granules) that sequester most
of the intracellular amine. The ability of the plasma-membrane transporters
to concentrate their substrates was not appreciated until platelet membrane
vesicles were shown to accumulate internal 5-HT to concentrations hun-
dreds of fold higher than the external medium when appropriate transmem-
brane ion gradients were imposed (26). These vesicle experiments
demonstrated conclusively that the plasma-membrane transporters gener-
ated gradients of their substrate amines, using the energy of transmembrane
Na
+
, Cl
–
,
and K
+
ion gradients.
3.1.1. Na
+
When a Na
+
concentration gradient (out > in) was imposed across the
vesicle membrane in the absence of other driving forces, this gradient was
sufficient to drive 5-HT accumulation (26). Coupling between Na
+
and 5-
HT transport follows from the fact that Na
+
can drive transport only if its
own gradient is dissipated. Thus, Na
+
influx must accompany 5-HT influx.
Na
+
-coupled 5-HT transport into membrane vesicles is insensitive to inhibi-
tors of other Na
+
transport processes such as ouabain and furosemide, sup-
porting the hypothesis that Na
+
and 5-HT fluxes are coupled directly by the
transporter(26,27). Many of these results have been reproduced in mem-
brane-vesicle systems from cultured rat basophilic leukemia cells (28),
mouse brain synaptosomes (29) , and human placenta (30).
3.1.2. Cl
–
The argument that Cl
–
is cotransported with 5-HT is somewhat less direct,
as it has been difficult to demonstrate 5-HT accumulation with only the Cl
–
gradient as a driving force. However, the transmembrane Cl
–
gradient influ-
ences 5-HT accumulation when a Na
+
gradient provides the driving force.
Thus, raising internal Cl
–
decreases the Cl
–
gradient, and inhibits 5-HT up-
take. External Cl
–
is required for 5-HT uptake, and Cl
-
can be replaced by
Br
–
; to a lesser extent, by SCN
–
, NO
3
–, and NO
2
–; and not at all by SO
4
2–,
PO
4
3–, and isethionate (27). In contrast, 5-HT efflux requires internal but
not external Cl
–
(27). The possibility that Cl
–
stimulated transport by electri-
cally compensating for electrogenic (charge moving) 5-HT transport was
ruled out by the observation that a valinomycin-mediated K
+
diffusion
potential (interior negative) was unable to eliminate the external Cl
–
require-
ment for 5-HT influx (27) .
3.1.3. K
+
The ability of internal K
+
to stimulate 5-HT transport was not immedi-
ately obvious, for two reasons. First, there was no absolute requirement for

30 Rudnick
K
+
in transport, and, second, no Na
+
cotransport system had ever been shown
to be coupled also to K
+
. Initially, we proposed that a membrane potential
generated by K
+
diffusion was responsible for driving electrogenic 5-HT
transport(26). However, subsequent studies showed that K
+
stimulated
transport even when the membrane potential was close to zero (11,31). In
the absence of a K
+
gradient, the addition of 30 mM K
+
simultaneously to
both the internal and external medium increased the transport rate 2.5-fold
(31). Moreover, hyperpolarization of the membrane by valinomycin in the
presence of a K
+
gradient had little or no effect on transport. Two conclu-
sions were drawn from these results. First, the transport process was likely
to be electrically silent. Second, since the K
+
gradient did not seem to act
indirectly through the membrane potential, it was likely to act directly
through exchange with 5-HT.
A study of the pH dependence of 5-HT transport revealed the reason that
5-HT transport still occurred in the absence of K
+
. A study of the pH depen-
dence of 5-HT transport. In the absence of K
+
, internal H
+
ions apparently
fulfill the requirement for a countertransported cation (12). Even when no
other driving forces were present (NaCl in=out, no K
+
present), a transmem-
brane pH difference (Δ pH, interior acid) could serve as the sole driving force
for transport. ΔpH-driven 5-HT accumulation required Na
+
and was blocked
by imipramine or by high K
+
(in=out), indicating that it was mediated by the
5-HT transporter, and not the result of non-ionic diffusion (12). From all of
these data, it was concluded that inwardly directed Na
+
and Cl
–
gradients,
and outwardly directed K
+
or H
+
gradients served as driving forces for 5-HT
transport (Fig. 2).
3.1.4. Electrical Consequences
Although studies using platelet plasma-membrane vesicles provided
direct evidence that 5-HT transport was electrically silent (11,31), evidence
relating the membrane potential to 5-HT transport has been mixed in other
systems. Bendahan and Kanner (28) found that 5-HT transport into plasma-
membrane vesicles from rat basophilic leukemia cells was stimulated by a
K
+
diffusion potential. However, other workers studying plasma-membrane
vesicles from mouse brain and human placenta concluded that 5-HT trans-
port in these tissues was not driven by a transmembrane electrical potential
(Δψ, interior negative) (32,33).
One might expect that electrogenicity could be easily tested if cells
expressing the 5-HT transporter could be directly impaled with microelec-
trodes. This has been done by Mager et al. (34), using Xenopus oocytes
injected with 5-HT transporter mRNA, with somewhat surprising results. A
simple prediction is that current should not flow across the membrane during

Biogenic Amine Transporters 31
5-HT transport if the transporter is electroneutral. In fact, a 5-HT-dependent
current has been measured, but closer inspection of its properties suggests that
it does not represent electrogenic 5-HT transport, but rather, a conductance
that is stimulated by transport. The key finding is that the transport-associated
current is voltage-dependent. Thus, the inward current increases as the inside
of the cell becomes more negative. In the same cells, however, [
3
H]5-HT trans-
port is independent of membrane potential. Therefore, it is very unlikely that
the voltage-dependent current represents the 5-HT transport process. Instead,
as discussed in Subheading 7, the current results from a newly discovered
ion-channel property of neurotransmitter transporters.
3.2. 5-HT Dopamine and Norepinephrine Transporters
use the Protonated Form of the Substrate
Because 5-HT, DA, and NE exist primarily in the protonated form at
physiological pH, many researchers have assumed that these substrates
are transported as cations. However, a small fraction of these amines exist
in the neutral or zwitterionic form at neutral pH, and it is important to
assess the possibility that these forms are the true substrates for transport.
In the case of the vesicular monoamine transporter (VMAT), the ionic form
of the substrate is a matter of some controversy. Different investigators have
reached opposite conclusions (35–37). If the neutral form were transported
by the plasma membrane biogenic amine transporters, one consequence
might be that the K
m for transport would be pH-dependent. As the pH
increases, the mole fraction of biogenic amine in the neutral or zwitterionic
form will increase sharply, but below pH 8.0–9.0, the majority of the sub-
Fig. 2. Driving forces for 5-HT transport. Na
+
and Cl
–
on the outside of the cell are
transported inward with the cationic form of 5-HT. In the same catalytic cycle, K
+
is
transported out from the cytoplasm. In the absence of internal K
+
, H
+
ions take the
place of K
+
. The energy released by downhill movement of Na
+
, Cl
–
, and K
+
provides
the driving force for 5-HT accumulation against its concentration gradient.

32 Rudnick
strate will be in the cationic form and the mole fraction of that form will not
change significantly. The K
m for total substrate (cationic and neutral) will
therefore appear to decrease if the neutral form is the substrate but will be
pH-independent if the cationic form is transported. Results with the 5-HT
and NE transporters (38,39) show no change in K
m with pH, and suggest
that the cationic form is the true substrate. An extensive study of dopamine
transporter concluded that the cationic (and possibly also the zwitterionic)
form of DA was the likely substrate (25) .
There is another consequence if the neutral or zwitterionic form is trans-
ported. The substrate would need to dissociate a H
+
ion before transport, and
then would bind H
+
after transport. By imposing a transmembrane pH dif-
ference (ΔpH), the equilibrium amine distribution across the membrane
could be influenced. In seeming agreement with this prediction, 5-HT accu-
mulation by platelet plasma-membrane vesicles is increased (in the absence
of K
+
) when the vesicle interior is acidified (12). However, this phenom-
enon represents the ability of H
+
to replace K
+
in 5-HT countertransport,
and not an influence on 5-HT protonation. In the presence of K
+
,ΔpH does
not stimulate 5-HT uptake, although this should occur if the neutral form of
5-HT is the true substrate (12).
3.3. Overall 5-HT Stoichiometry
The number of Na
+
, Cl
–
, and K
+
ions transported with 5-HT transporter
has been estimated by imposing known Na
+
, Cl
–
, and K
+
concentration gra-
dients across the plasma membrane as a driving force, and measuring the 5-
HT concentration gradient accumulated in response to that driving force at
equilibrium. This is essentially a thermodynamic measurement, balancing a
known driving force against a measured gradient of substrate. Technically,
such measurements require that the imposed ion gradients are relatively
stable, so that the available driving force is known at a given time after
imposition of the ion gradient.
3.4. Transport Kinetics can Suggest, but not Determine,
Stoichiometry
Kinetic techniques have also been used to assess the Na
+
, Cl
–
, and K
+
stoichiometry for 5-HT, NE, and DA transport. One technically simple tech-
nique is to measure the dependence of transport rate (or its kinetic determi-
nantsK
m or V
max) on Na
+
, Cl
–
, or K
+
concentration, and to calculate a Hill
coefficient for that ion. Using this analysis for the 5-HT and NE transporters
yields an n of one for both Na
+
and Cl
–
in membrane vesicles (40–42), where
initial rates of transport showed a simple hyperbolic dependence on Na
+
or
Cl
–
—consistent with a Na
+
:Cl
–
:substrate stoichiometry of 1:1:1.

Biogenic Amine Transporters 33
However, steady-state kinetics do not necessarily provide accurate infor-
mation on cotransport stoichiometry (43) . It is possible that more than one
Na
+
ion is required for substrate binding or even translocation (as reflected
in the Hill coefficient calculated from rate measurements), but that only one
of those Na
+
ions is actually cotransported. It is also possible that a substrate
is cotransported with two Na
+
ions, but that the affinities or rates of associa-
tion of the two Na
+
ions are so disparate that the initial rate of transport is
dependent on only the weaker binding or slower associating of the two, lead-
ing to an apparent Hill coefficient of 1. These difficulties are inherent in any
kinetic method, whether transport is measured directly by tracer flux, or
indirectly by measurements of electrical currents that may accompany trans-
port. Thus, the dependence of transport rate on the concentration of a given
ion may suggest a transport stoichiometry, but cannot provide proof for it.
A still more direct method is to measure the flux of driving ions as well as
the flux of substrate. Usually the basal levels of ion fluxes are too fast rela-
tive to the rates of substrate transport, but in a purified, reconstituted sys-
tem, Kanner and colleagues were able to measure Na
+
and Cl
–
flux along
with GABA flux by the GABA transporter. In this case, in which both ther-
modynamic and direct kinetic data exist, both methods indicate a Na
+
:Cl
–
:GABA stoichiometry of 2:1:1 (63, 39, 40).
3.5. Thermodynamic Approach
Because kinetic approaches may be experimentally difficult or mislead-
ing, it is essential to confirm the stoichiometry by a thermodynamic mea-
surement. In the thermodynamic method, known Na
+
, Cl
–
, or K
+
concentration gradients are imposed across the plasma membrane as a driv-
ing force, and the substrate concentration gradient in equilibrium with that
driving force is measured. By varying the concentration gradient of the driv-
ing ion, and measuring the effect on substrate accumulation, the stoichiom-
etry can be calculated. For a simple system in which two solutes, A and B,
are cotransported, a plot of ln(A
in/A
out) vs ln(B
out/B
in) gives the B:A stoichi-
ometry as its slope. As a special case, if the stoichiometry is 1:1, then a plot
of A
in/A
out vs B
in/B
out will be a straight line. Using this method, a 1:1 cou-
pling was determined for 5-HT transport with both Na
+
(18) and K
+
(26).
The Cl
–
stoichiometry was deduced from the fact that 5-HT transport was
not affected by imposition of a Δψ (interior negative), and was therefore
likely to be electroneutral. Because 5-HT is transported in its cationic form
(12,39), only a 5-HT
+
:Na
+
:Cl
–
:K
+
stoichiometry of 1:1:1:1 is consistent with
all the known facts. Obviously, this analysis requires an experimental sys-
tem such as membrane vesicles, where the composition of both internal and
external media can be controlled. This method also relies on the ability to

34 Rudnick
measure, or at least to estimate, an equilibrium substrate gradient under con-
ditions in which the ion gradients are known (43).
3.6. Each Transporter has a Characteristic Coupling of Ion Flux
to Substrate Flux
3.6.1. Norepinephrine Transport
Although no membrane-vesicle systems containing the DA transporter
have been described, two plasma-membrane vesicle systems have emerged
for studying NE transport: the placental-brush-border membrane (42) and
cultured PC-12 cells (47). Harder and Bonisch (47) concluded that NE trans-
port into PC12 vesicles was coupled to Na
+
and Cl
–
and was electrogenic,
but they failed to arrive at a definitive coupling stoichiometry because of
uncertainties about the role of K
+
. According to their analysis, stimulation
of NE influx by internal K
+
resulted either from direct K
+
countertransport
as occurrs with SERT (31), or from a K
+
diffusion potential which drives
electrogenic NE influx, as with GAT-1 (48) . Ganapathy and colleagues (42)
studied NET-mediated transport of both NE and DA into placental-mem-
brane vesicles (DA is utilized by NET as a substrate) (49). They reached
similar conclusions regarding ion coupling, but also were left with some
ambiguity regarding K
+
. In fact, the effects of ions on NET-mediated DA
accumulation were similar to those observed with SERT-mediated 5-HT
transport in the same membranes, and the two activities were distinguished
only by their inhibitor sensitivities (19). Part of the difficulty in interpreting
and comparing these data stems from the fact that they were obtained in
various cell types, with unknown and potentially very different conductances
to K
+
.
Two further problems made it difficult to interpret previous data on NET
ion coupling. Both previous studies assumed that the cationic form of the
catecholamine substrate was transported (42,47), and did not consider the
possibility that the neutral or zwitterionic form was the true substrate. More-
over, previous studies estimated NET stoichiometry using kinetic rather than
thermodynamic measurements. The number of Na
+
ions cotransported with
each catecholamine substrate was estimated from the dependence of trans-
port rate on Na
+
concentration (42,47).
The author and colleagues established LLC-PK
1 cell lines stably express-
ing the biogenic amine transporters SERT, NET, and DAT, as well as the
GABA transporter GAT-1. Using these cell lines, we characterized and com-
pared the transporters under the same conditions and in the same cellular
environment(49). One attractive advantage of LLC-PK
1 cells is that it has
been possible to prepare plasma-membrane vesicles that are suitable for
transport studies (50). We took advantage of this property to prepare mem-

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‘I know what you did,’ said Mrs. Hayward, with a smile. ‘You said, I
wish Elizabeth were here. And she heard it, and suggested that you send for
me. Most likely she was a little amused about Elizabeth. I know your way,
and what the young fellows say, that you always want Elizabeth, whatever
happens.’
‘So I do—so I do; though I can’t tell how they know, the jackanapes.
Here we are at the door.’
‘You must smuggle me upstairs before anybody sees me, for I’m very
untidy; and I know how fresh they will all look in their morning things,’
cried Mrs. Hayward, with a shade of disquietude in her eyes.
‘Oh yes, you shall be smuggled upstairs,’ cried the Colonel, confident in
the security of the early hour. And presently the pair found themselves in
the cheerful room prepared for the newcomer, with tea set out upon a table.
Elizabeth took at once the command of the position. She gave him some
tea, then dismissed him to an easy chair in his own room, which
communicated with hers, where, as he began to doze, he could see her little
figure moving about, appearing and disappearing, as she unpacked her
things and made herself comfortable. She looked, he thought, as if she had
been there all her life. It was a faculty peculiar to her. She made the barest
barrack-room look like herself somehow, before she had been half an hour
in it. Wherever she was, the place began to appear like home directly. He
had the immense sense of relief which a man in charge of a difficult post
feels on the arrival of his commanding officer who takes over the
responsibility, and that delightful loosening of moral tension filled him with
pleasant drowsiness. His eyes, half shut, half open, were conscious of her,
and that everything was being looked after; and, as a matter of fact, he had
not slept well for two or three nights, though Elizabeth had scoffed at this.
He had a most refreshing doze while she dressed and made herself look as
fresh as the morning. As for her having been untidy, even after the night-
journey, that was a thing impossible to Elizabeth. But he knew that she
would come out looking fresher than the day.
She was a little woman of about forty-five, with the complexion of a girl,
and eyes that were as blue as an infant’s, but with the quality of brightness
which belongs more frequently to a darker hue. Not soft and dreamy as blue
eyes should be, but keen and clear, dancing with light—eyes which saw
behind as well as before, and which nothing could elude. There was no

sleep or weariness in them, but there was, visible to her own perception as
she looked at herself in the glass, a keener glitter of uneasiness, a little
curve of anxiety in the lids. He seemed to think only of this possible
revelation of the past—which, no doubt, was important, very important; but
of the future, which she saw so distinctly opening upon them, a future
entirely new, distracting, for which neither she nor he had any preparation,
he seemed to take no thought. That was Henry’s way, she said to herself, to
be overwhelmed by one view of a question, which had half a dozen other
aspects more important, and to make himself quite comfortable about it
when the first shock was over, without an idea of what the consequences
might be: dear old stupid that he was! She, too, glanced at him as she
passed and repassed the doorway, with a tenderness in which there was a
mixture of amusement and partial irritation and fun and sympathy, all
mingled together. His goodness, his strength, his helplessness and confusion
of mind, his high courage and authority and judgment, and his complete
dependence and docility, were all so evident to those keen eyes of hers,
which adored him, laughed at him, smote him with keen shafts of criticism,
made haloes of glory about him all at one and the same moment. He had
brought her many a ravelled skein to disentangle, but never any so serious
as this. Joyce dead had been a shadow often discouraging upon her life, but
Joyce living filled her lively soul with a shrinking of dismay. And of this he
did not seem to have a thought.

CHAPTER VII
Janet Matheson was busy with her broth, which was boiling softly, slowly
over the fire, ready to receive the vegetables—red, white, and green—the
carrots and turnips and early crisp cabbage, all nicely cut and glistening
with freshness and cleanness, which she had just prepared to add to the
contents of the pot. She had a large brown holland apron covering her
cotton gown, and a thick white cap surrounding her frosty-apple cheeks.
The room was as neat and bright as her own little active figure. The little
greenish window behind was open to admit the scent of the mignonette in
the garden, and the pale pink monthly rose which looked in. On the sill of
the opened window there was a line of books, and a writing-table stood
under it, slightly inappropriate, yet disturbing nothing of the homely
harmony of the cottage. The door to the street was open too, and any
passing stranger could have seen Janet, who now and then looked out, with
a carrot in one hand, and the knife with which she was scraping it in the
other, wondering where that lassie J’yce could have gone to. The holidays
had begun, and Joyce was free. She had done her share of the household
service before she went out; but her tender old guardian was of opinion that
about this hour ‘a piece’ was essential, though that was a thing of which
Joyce could never be got to take proper heed. She had turned her back to
the world, however, and was emptying her bowlful of vegetables into the
pot, when Mrs. Hayward tapped at the open door. Janet said mechanically,
‘Come in—come away in’ without hurrying the operation in which she was
engaged. When she turned she found another bright-eyed woman looking in
at her from the pavement.
‘May I come in?’ said Mrs. Hayward.
‘Certainly, mem, ye may come in, and welcome. Come away,’ said Janet,
lifting a wooden chair, and placing it, though the day was very warm,
within reach of the fire. It was clean as scrubbing could make it, yet she
dusted it mechanically with her apron, as is the cottager’s use. Mrs.
Hayward watched every movement with her bright eyes, and observed all
the details of the little house. A simple woman, looking like a French
peasant with her thick cap; a little rustic village house, without the slightest
pretension of anything more. And this was the house in which the girl had

been bred who Henry said was a lady—a lady! He knew so little, poor
fellow, and men are taken in so easily. No doubt she was dressed in cheap
finery, like so many of the village girls.
‘I wanted, if you will allow me, to make some inquiries about your—but
she is not your daughter?’
‘About Joyce?’ said the old woman quickly. She put down the bowl and
came forward a few steps, from henceforward departing from her rôle of
simple hospitality and friendliness, and becoming at once one of the parties
to a duel, watching every step her adversary made. ‘And what will ye be
wanting with Joyce?’ she asked, planting her foot firmly on the floor of her
little kingdom. She was queen and mistress there, let the other be what she
might.
‘It is difficult to say it in a few words,’ said Mrs. Hayward. ‘I have heard
that though you have brought her up like your child, and been so tender to
her, yet that she is no relation of yours.’
‘There are idle folk in every place,’ said Janet sententiously, ‘who have
nothing to do but to stir up a’ the idle tales that ever were heard about the
country-side.’
‘Do you mean, then, that this is an idle tale?’
The two antagonists watched each other with keen observation, and
Janet saw that there was something like pleasure, or at least relief, in her
adversary’s manner of putting the question. ‘It a’ depends on the sense it’s
put in,’ she said.
‘We can’t go on fencing like this all day,’ cried Mrs. Hayward quickly. ‘I
will tell you plainly what I want. My husband has seen the girl whom you
call Joyce.’
‘Mem, you might keep a more civil tongue in your head,’ said Janet,
‘and ca’ her something else than the girl.’
‘What should I call her? I have not seen her. It is not with any will of my
own that I am here. I hear her very highly spoken of, and your great
kindness to her, and her—what is far more uncommon—gratitude to you.’
‘Mem,’ said Janet, ‘we Scots folk, we’re awfu’ unregenerate in the way
of pride. We are little used to have leddies coming inquiring into our maist
private concerns, ca’ing a woman’s affection for her bairn kindness, and a
good lassie’s good heart for her faither and mither gratitude.’

‘I quite agree with you,’ said Mrs. Hayward, rising up suddenly and
putting out her hand. ‘You are quite right, and I am—unregenerate as you
say. The reason is, I have been a little put out this morning, and I have
inquiries to make which I don’t make with any heart. I have come to ask
you to let me see the things which Joyce’s mother left behind her—or at
least the letters which Mrs. Bellendean told my husband of. A glance at
them would possibly settle the question. My husband thinks—that he knows
who she is.’
Janet had wiped her hand with her apron, and given it to her visitor, but
with some reluctance. ‘And wha may your husband be, mem?’ she said.
‘He says he spoke to you the other day. He is, though I say it, a
distinguished soldier. He is Colonel Hayward, who was Captain
Bellendean’s commanding officer.’
Janet was not greatly moved by Colonel Hayward’s distinction, nor by
his grade, but that he should be the Captain’s commanding officer
impressed her at once. ‘Then he’ll be a gentleman that’s far aboon the like
of us,’ she said, ‘and no’ a man that would put forth his hand for naught, or
disturb a decent poor family without just cause.’ She stood a little, fingering
her apron, ‘glowering frae her,’ as she would have said, casting a wistful
look into vacancy. ‘It will maybe be something—that would make a great
change,’ she said, her lips quivering a little, ‘if it cam’ true.’
‘I am afraid it would make a great change,’ said Mrs. Hayward, and she
added with a sigh, ‘both to you and to me.’
‘To you!’ Janet clasped her hands. ‘What will you have to do with it?
What would it be to the like of you? You’re no—you’re no——? or the
Cornel——?’ The old woman put her hand with natural eloquence to her
breast. ‘My heart’s just louping like to choke me. Oh mem, what would it
be to you?’
‘Look here,’ said her visitor. ‘We may be giving ourselves a great deal of
unnecessary trouble. It may happen that when I see the letters it will all
come to nothing. Then let me see them directly, there’s a dear woman. That
is the best and the only thing to do.’
There was a sweep of energetic movement about this rapid little lady that
pressed forward Janet’s reluctant feet. She took a step or two forward
towards the stair. But there she paused again. ‘I’ve aye said to Peter we
must keep a loose grip,’ she said. ‘And when she was only a wean it would

have been nothing: but she’s come to be that between him and me, that I
canna tell how we’re ever to part. I’ve never said it to her. Na. I’m no’ one
to spoil a young cratur’ with praisin’ her. I’ve kept it before her, that if she
had mair headpiece than the rest, it was nae credit of hers, but just her
Maker that had made her sae. It’s no’ for that. It’s no because she’s an
honour and a glory to them that have brought her up. Whiles the one that ye
are proudest of is just the one that will rend your heart. But she’s that sweet
—and that bonnie—bonnie in a’ her ways—ye canna help but see she’s a
leddy born; but to take upon hersel’ because o’ that. Na, na. That shows ye
dinna ken our J’yce. Oh, I aye said haud a loose grip!’ cried the old woman,
with broken sobs interrupting her speech. ‘I’ve said it to my man a thoosan’
times and a thoosan’ to that; but it’s mair than I have done mysel’ at the
hinder end.’
The stranger’s bright eyes grew dim. She put her hand on Janet’s arm. ‘I
should like to cry too,’ she said—‘not like you, for love, but for pure
contrariness, and spite, and malice, and all that’s wicked. Come and show
me the letters. Perhaps we are just troubling ourselves in vain, both you and
I——’
‘Na, na, it’s no’ in vain,’ said Janet, restraining herself with a vehement
effort. ‘If it may be sae this time, it’ll no’ be sae anither time. We may just
be thankful we have keepit her sae lang. I never looked for it, for my pairt.
I’ll gang first, mem, though it’s no’ mainners, to show you the way. This is
her cha’amer, my bonnie darling; no’ much of a place for a leddy like you
to come in to, or for a leddy like her—God bless her!—to sleep in. But we
gave her what we had. We could do nae mair—if ye were a queen ye could
do nae mair. And she’s been as content all her bonnie days as if she was in
the king’s palace. Oh, but she’s been content; singing about the house that it
was a pleasure to hear her, and never thinking shame—never, never—of her
auld granny, wherever she was. She has ca’ed me aye granny—it was mair
natural; and nae slight upon the poor bonny bit thing that is dead and gone.’
Janet went on talking as she placed a chair for the visitor, and went
forward to the rude little desk where Joyce kept her treasures. She talked
on, finding a relief in it, a necessity for exertion. Mrs. Hayward looked
round the little homely place, meanwhile, with a curiosity which was almost
painful. It was a tiny little room with a sloping roof, furnished in the
simplest way, though a white counterpane on the little bed, and the white
covering of the little dressing-table in the window, gave an air of care and

daintiness amid the simple surroundings. A few photographs of pictures
were pinned against the wall. But the place of honour was given to two
photographic groups framed, one representing a group of school children,
the other a band of (Mrs. Hayward thought) very uncouth and clumsy
young men. Janet, with a wave of her hand towards these, said— ‘Hersel’
and her lassies,’ and ‘Andrew and some of his freends.’ It seemed to the
keen but agitated observer, in the formality of the heavy cluster of faces, as
if all were equally commonplace and uninteresting. She sat down and
watched, with an impatience which nothing but long practice could have
kept within bounds, while Janet opened the desk which stood against the
wall, and then a drawer in it, out of which at last, with trembling hands, she
brought a little parcel, wrapped in a white handkerchief. Janet was as
reluctant as her visitor was eager. She would fain have deferred the test, or
put it aside altogether. Why had she kept these papers for her own undoing?
She undid the handkerchief slowly. There fell out of it as she unfolded it
several small articles, each done up in a little separate packet. ‘A’ her bit
things that she had,’ Janet explained. ‘A locket round her neck, and a bit
little watch that winna go, and the chain to it, and twa rings. I wanted Joyce
to wear them, but she will wear nothing o’ the kind, no’ so much as a bit
brooch. Maybe you will ken the rings if you see them,’ said Janet, always
anxious to postpone the final question, putting down the larger packet, and
picking up with shaking fingers, which dropped them two or three times
before they were finally secured, the tiny parcel in which the ornaments
were enclosed.
‘No, no,’ said Mrs. Hayward. ‘The letters are the only things. Show me
the letters, I implore you, and don’t let us torture ourselves with suspense.’
‘Ae kind of torture is just as bad as another,’ said the old woman,
undoing with great unsteadiness the cotton-wool in which the trinkets were
enclosed. She held them out in the palm of her brown and work-scarred
hand. A little ring of pearl and turquoise, made for a very slender finger, in a
simple pattern, like a girl’s first ornament, and beside it another, equally
small, a ruby set round with brilliants. The glimmer of the stones in the old
woman’s tremulous hand, the presence of these fragile symbols of a life and
history past, gave the spectator a shock of sympathetic pain almost in spite
of herself. She put them away with a hurried gesture— ‘No, no; nothing but
the letters. I never saw these before; I know nothing—nothing but the
letters. Show me the letters.’

Janet looked at the trinkets and then at Mrs. Hayward, with a rising light
of hope in her eyes. ‘Ye never saw them before? It will just be somebody
else and no her ye was thinking of? That’s maist likely, that’s real likely
——’ wrapping them up again slowly in their cotton-wool. Her fingers,
unused to delicate uses, were more than ever awkward in their tremor. To
put them back again was the business of several minutes, during which she
went on: ‘You will not be heeding to see the other things? I have them here
in her box, just as she left them—for Joyce would never hear of puttin’ on
onything—and they’re auld-fashioned, nae doubt, poor things. You’ll no be
heeding?—oh ay, the letters—I’m forgetting the letters. But, mem, if ye’ve
nae knowledge of her bit rings and things, ye will get nothing out of the
letters. There’s nae information in them. I’ve read them mysel’ till I could
near say them off by heart, but head or tail of them I could mak’ nane. Here
they are, any way. She’s made a kind of a pocket-book to put them in—a’
her ain work, and bonnie work it is—flowered with gold; I never kent
where she got the gift o’t. Ye would think she could just do onything she
turned her hand to. Ay, there they are.’
And with no longer any possible pretence for delay, she thrust a little
velvet case into Mrs. Hayward’s hand—who between impatience and
suspense was as much excited as herself. It was worked in gold thread with
a runic cross, twisted with many knots and intertwinings, and executed with
all the imperfections of an art as uninstructed as that of the early workers in
stone who had wrought Joyce’s model. Inside, wrapped carefully in paper,
were the two silent witnesses—the records of the tragedy, the evidence
which would be conclusive. Mrs. Hayward’s hands trembled too as she
came to this decisive point—they dropped out of her fingers into her lap.
Her heart gave a leap of relief when her eye fell on the handwriting of the
uppermost, which was unknown to her. The other was folded, nothing
showing but the paper, yellow and worn at the edges with much perusal. In
spite of herself, she took this up with a feeling of repugnance and dread—
afraid of it, afraid to touch it, afraid to see—— what instinct told her must
be there. She paused, holding it in her hand, and gave Janet a look. No
words passed between them, but for the moment their hearts were one.
Mrs. Hayward opened the folded paper, then gave a low cry, and looked
at Janet once more—and to both the women there was a moment during
which the solid earth, and this little prosaic spot on it, seemed to go round
and round.

‘It will be what you was looking for?’ said Janet at last. She had been
full of lamentation and resistance before. She felt nothing now except the
hand of fate. The other shook her head.
‘Yes,’ she replied, and said no more.

CHAPTER VIII
In the meantime Colonel Hayward was walking up and down the village
street, waiting for his wife. He passed and repassed the door two or three
times. He was very nervous, hanging about, not knowing what to make of
himself. The church stood at the end of the street, and a path led down by
the side of the churchyard, in the direction of Bellendean. As he came to the
end of this, he stopped in the abstraction of his mind to look down the line
of shade which a high hedgerow opposite to the low mossy wall of the
churchyard threw half-way across the path. Some one was coming along in
this clear and soft shadow, which was so grateful in the midst of the
sunshine. It startled him to see it was Joyce, in her dark dress, her face
relieved against the broad brim of an untrimmed straw hat, which added in
its tone of creamy white additional force to the very delicate tints of her
face, so clear in the shadowy air, with an impression of coolness in the
midst of great warmth. He cast an anxious look of suspense over his
shoulder towards the house where his wife was; but as he did not see her,
nor any sign of her coming, he turned down the path to meet Joyce. It was
rather by way of diverting his own anxiety than from any eagerness to
address her. He seemed to want somebody to whom he could talk to relieve
his own mind; for up to this moment, except from curiosity and anxiety in
respect to the past, and a certain admiration of herself and her demeanour, it
had not been Joyce, upon her own account, who had interested the Colonel.
He had not had leisure as yet to get so far as her—for herself. He went on to
talk to her because she was in it, concerned like himself, though she might
not be aware of the fact, in the matter which his wife at present was
engaged in clearing up. It was as if the scene then going on at the cottage
was a consultation of doctors upon the life or death of a beloved patient.
Those who are waiting breathless for the opinion, which is at the same time
a sentence, are glad to get together to ask each other what they think,—at
least, to stand together and wait, feeling the support of company. This was
Colonel Hayward’s feeling. He went towards the girl with a sense that she
had more to do with it than any one else—but not with any perception of its
immense importance to her.

Joyce had gone out in the freedom which comes to all the members of
the scholastic profession, small and great, with the first morning of the
holidays. To have no lessons to give, no claim of one kind or another,
nothing but their own occupations, whatever they may be, gives to these
happy people a sense of legitimate repose. For one thing, the members of
almost every other profession have to go away to secure this much-desired
leisure, but to the teacher it comes, without any effort, by appointment of
nature, so to speak, by a beneficent arrangement which takes all selfishness
out of the enjoyment, since it has been invented, not for the good primarily
of himself, but of the flock who are so happily got rid of, to their own
perfect satisfaction. The sweet consciousness that the happiness and
freedom of so many sufferers have been consulted before one’s own, gives
sweetness and grace to it. Joyce had risen this morning with that exquisite
sense of freedom, and she had gone out with a book as soon as the
household work she never neglected was over, to read and muse on a
favourite spot, a point in the park at Bellendean out of reach of the house,
where behind a great screen of trees the wayfarer came suddenly in sight of
the Firth, the circle of low hills which protects the narrower sea at the
Queen’s Ferry, and the sheltered basin of St. Margaret’s Hope. The sight of
this wonderful combination of sea and sky and solid soil, the soft hills
rising round, the mass of grey stones on the water’s edge, which marks a
ruined castle, the island in the midst, the widening out beyond into the
infinite, into the wider Firth and the stormy waters of the northern sea,
affording an ever-open door for the fancy,—were delightful to this
imaginative girl. She had taken her book, but she did not open it—for which
she upbraided herself, confessing in the secret depths of her soul that
Andrew would not have done so,—that he would have read and expounded
and discussed and found a new beauty in every line, where she, so much his
intellectual inferior, did nothing. She did not even think—if further avowal
must be made, she did not even see the lovely landscape for the sake of
which she had come here. It entered into her, reflecting itself in her dreamy
eyes, and printing itself in her mind; but she did not look as Andrew would
have done, finding out beautiful ‘lights,’ and commanding all the details of
the scene. Joyce was a little short-sighted, and did not see the details. It was
to her a large blurred celestial world of beauty and colour, and abundant
delicious air and sunshine. Her thoughts went from her, where she sat in the

heart of the morning, looking over the Firth, with all its breadth of melting
light and reflection, to those low hills of the farther shore.
It had been thus that she had entered upon her holidays in the other days
when life had no cares. The dreamings about Lady Joyce, and all the
speculations as to her future, had come in other scenes, where there was a
want of brightness and of a stronghold of her own to retire into. Here she
had not needed that fanciful world of her own. But to-day Joyce was in a
different mood. After a while she began to become insensible altogether to
the scene, and resumed more personal musings instead. ‘Young lady, where
did you get your name?’ It was not the first time she had been so
questioned. Half the people she met asked her the same: but not as Colonel
Hayward did. ‘I knew some one once’—what did he mean? why did he not
come back and tell her? These thoughts became urgent after a while, so that
she could not sit and dream, as was her wont in her favourite spot. She got
up with a little impatience and vexation and disappointment to return home.
But in the lane which led up to the village street, in the clear shadow of the
tall hawthorn hedge, behold some one advancing to meet her, at sight of
whom her heart began to beat—more loudly than it had ever beaten at the
sight of Andrew Halliday; it sprang up thumping and resounding. ‘He
knows who I am,’ she said to herself. ‘Perhaps he will tell me; perhaps he is
looking for me to tell me. Perhaps he is something to me.’ Her veins seemed
suddenly to fill with a rushing quick-flowing stream.
Colonel Hayward took off his hat as he came up. This was to him an
everyday action, but to her an unusual grace, a homage which only lately
had ever been given to her, and which she esteemed disproportionately as a
sign of special chivalry. It brought the colour to her cheeks, which ebbed
again the moment after in the fluctuations of her anxiety. The old Colonel
looked very anxious too; his face was agitated, and paler than usual. When
he came up to her he stopped. ‘I don’t think,’ he said, ‘that we were ever
introduced to each other; but still—— You have been taking a walk this fine
morning?’
‘The holidays have just begun, sir,’ said Joyce respectfully. ‘This is the
first day: and though I am very fond of my work, freedom is sweet at first.’
‘Only at first?’
‘It is always sweet,’ she said, with a smile; ‘but never so delicious as the
first day.’

Their hearts were not in this light talk, and here it came to an end. He
had turned with her, and they were walking along side by side. Great
anxiety—tremulous and breathless suspense—were in the minds of both on
the same subject—and yet they regarded it in aspects so different! The soft
transparent shadow of the hedge kept them from all the flicker of light and
movement outside, giving a sort of recueillement, a calm of gravity and
stillness, to the two figures. Had they been in a picture, there could have
been no better title for it than ‘The Telling of the Secret.’ But yet there was
no secret told. He was absorbed in his own thoughts, and unconscious of the
wistful looks which she gave him timidly from time to time. At last he
turned upon her, and asked the strangest question, with a tremor and quiver
in all his big frame.
‘Do you remember your mother?’ he said.
‘My mother!’ The sudden shock brought a wave of colour over her. ‘Oh,
sir,’ said Joyce, ‘how could I remember her? for she died when I was born.’
‘True, true—I had forgotten that,’ he said, with an air of confusion. Then
added— ‘You must forgive me. My mind was full——’
Of what was his mind full? He fell silent after this, and for some time no
more was said. But it gradually came to be impossible to Joyce to keep
silence. She turned to him, scarcely seeing him in the rush of blood that
went to her head.
‘Did you know my mother?’ she said. ‘Oh, sir, will you tell me? Do you
know who she was?’
‘I can’t tell—I can’t tell,’ he said, shaking his head. ‘It may be all a
mistake. We must not make too sure.’
‘Then you think——’ she cried, and stopped, and looked at him,
searching his face for his meaning—the anxious open face which was held
before her like a book—though he did not look at her in return. She put her
hand, with a light momentary touch, on his arm. ‘Perhaps you don’t know,’
she said hurriedly, ‘that I have things of hers—things she left—that would
settle it—that would show you——’
He made a little gesture of assent, waving his hand. ‘My wife is there:
that is what keeps me in this suspense.’
‘Where? Where?’

He pointed vaguely in the direction of Joyce’s home. ‘She has gone—to
see everything,’ he said.
For the moment a flash of sudden anger came to the eyes of Joyce. ‘They
are all mine!’ she cried. ‘It was to me she ought to have come. I am the one
chiefly concerned!’ Then the flash quenched itself, and her look grew soft
and wistful once more. ‘Oh, sir,’ she said, ‘if it was the Joyce you thought—
if it was her you supposed—who was she? To tell me that, even if it should
turn out all different, would do no harm.’
‘It would do no good either,’ he said: then turned round to her, and took
her hand between his two large brown hands, which were trembling. ‘You
are very like her,’ he said—‘so like her that I am forced to believe. She
looked just as you are doing when I saw her last. Some relationship there
must be—there must be!’ Here he dropped her hand again, as if he had not
known that he held it. ‘There was wrong done to her—the Joyce I mean.
She was made very unhappy; but no wrong was meant on—on my—on—
on his part. Would you really like to hear the story? But it may turn out to
be nothing—to have nothing to do with you.’
‘Oh, tell me; it will fill up the time; it will ease the suspense.’
‘That is what I feel,’ he said; ‘and you will keep the secret—that is, there
is no secret; it is only what happened to—— what happened long, long ago
—to—to one of my friends: you understand,’ he said tremulously, but with
an effort to be very firm, looking at her, ‘to—one of my friends.’
Joyce made a sign of assent, too much absorbed in what she was about to
hear to think what this warmth of asseveration meant. It was a relief to him
to speak. It was like going over all the changes of the illness when a
beloved sufferer lies between life and death.
‘They met,’ he said, ‘abroad, at a foreign station. She was very young.
She was with people that were not kind to her. They married in a great
hurry, without proper precautions, without thinking that anything could be
wrong. They came home soon after for her health, and I—I had to—I—I
don’t quite remember——’ his voice seemed to die away in his throat; then
with another effort he recovered it and went on— ‘Her husband had to
leave her and go back—to his duty: and then she heard from some wicked
person—oh, some wicked person!—God forgive her, for I can’t—that it
was not a true marriage. It was, it was! I protest to you no thought of harm

—good Lord! nothing but love, honest love—and it was all right, all right,
as it turned out.’
‘But she thought—she had been deceived!’ Joyce listened with her head
drooping, keeping down the climbing sorrow in her throat, hardly able to
find her voice.
‘She was always hasty,’ he said. ‘I am not the one to blame her—oh no,
no—it was not wonderful, perhaps, that she should believe. And letters to
India were not then as now—they took so long a time; and something
happened to delay the answer. It was what you call nobody’s fault—only an
accident—an accident that cost——’
‘You are very, very kind—oh, you are kind; you speak as if you had felt
for her with all your heart—as if she had been your very own.’
He gave her a startled look, and made a momentary pause: then he
proceeded, ‘That’s all,—all that anybody has known. She disappeared. His
letter came back to him. He could not get home to search for her. It had to
be trusted to others. After years, when I came back, I—I—but nothing could
ever be found.’
‘Sir,’ said Joyce, gasping a little to keep down her sobs, ‘I think that
must have been my mother. I—think it must be. She begins in her letter to
tell him—she calls him Henry—was that his name?’
The old Colonel made a noise in his throat which sounded like a sob too:
he nodded his head in assent, as if he could not speak.
‘She begins to tell him—is he living still?’
This question had the strangest effect upon Colonel Hayward. He turned
round upon her, steadying himself, looking her in the face, with momentary
wonder and something like indignation: then the energy died out of him all
at once, and he nodded his head again.
‘My father! then I have a father,’ said Joyce, with a voice as soft and
tender as a dove’s. She was not now paying any attention to him or his
looks, but was entirely absorbed in this new wonderful discovery of her
own.
But he started with a sudden cry— ‘Good God!’ as if something new—
something too astounding to understand—had flashed upon him. Her
father! why, so it was!—so he was—— He had thought of no subject but
this for days, and yet this point of view had not opened upon him. They had

reached the head of the lane, and were now in the village street, turned
towards the cottage in which Joyce had lived all her life, and near enough to
see the light little figure of Mrs. Hayward standing at the door. This caught
his attention, but not hers. For Joyce had plunged suddenly with a new
impulse back into the enchanted country of her dreams. A father—and one
who had done no wrong—who was not to blame—a living father! It was
only when she turned to Colonel Hayward, after the first bound of
exhilaration and breathless pleasure, to ask him, clasping her hands
unconsciously, ‘Who is my father?’ that she saw the extraordinary
commotion in his face. He was looking at her, and yet his eyes made quick
voyages to and from his wife. The lines of his face had all melted into what
Joyce felt to be the ‘kindest’ look she had ever met. And yet there was alarm
and boundless anxiety in it. He looked as if he did not hear her question, but
suddenly laid his hand upon hers, and gave it a strong momentary pressure.
‘I must know first. I must speak to my wife,’ he said incoherently. ‘God
bless you!—I must ask Elizabeth. You must wait: I must speak to Elizabeth.
But God bless you, my dear!’
He was already gone, hastening with long steps up the street. The
thought passed through Joyce’s mind that this must have been a dear friend,
—some one, perhaps, who had loved her mother: and a man with the
tenderest heart. There was something in his ‘God bless you’ which seemed
to fall upon her like the dew—a true blessing; the blessing of one who had
always been her friend, though she had never known him. She did not hurry
to follow him to satisfy herself, but went on quietly at her usual pace,
looking at the old gentleman’s long swift steps, and thinking of a camel
going over the ground. He was from the East, too; and he devoured the way,
hastening to the little figure which had perceived and which was waiting for
him. Joyce had the faculty of youth to remark all this, yet keep up her own
thoughts at the same time. She saw old Janet standing at the door looking
out, with the hem of her apron in her hand, which was her gesture when her
mind was much occupied or troubled: and the little lady in the street
standing waiting, and then, her own old friend, the Colonel, hurrying up,
putting his arm within the lady’s, leading her away with his head bent over
her. There was a certain amusement in it all, which floated on the surface of
the great excitement and wonder and delight of the discovery she had made.
A father; and a dear old friend, the kindest, the most sympathetic, who

blessed her, and who had a right to bless her, having loved (she could not
doubt it) her mother before her.
Joyce did not know what the next disclosure might be,—did not think for
the moment that, whatever it was, it must change the whole tenor of her life.
Nor did she think that there was still a doubt in it,—that it might yet come
to nothing, as he had said. Oh no, it could not come to nothing; everything
pieced in to the story. The doubt with which Janet had always chilled her,
that a young creature disappearing so utterly, with no one to care for her, no
one to inquire after her, must have had a story in which shame was involved
—how completely was it dissipated and explained by this real tale! Oh, no
shame! she had felt sure there could not be shame—nothing but the cruel
distance, the fatal accident that had delayed the letter, those strange
elements of uncertainty which mix in every mortal story, which (Joyce
remembered from that reading which had hitherto been her life) the ancients
called fate. And what could they be called but fate? If it had come in time
that letter! as letters which mean nothing, which are of no consequence,
come every day—and yet he had said the delay was nobody’s fault. Was it
less fatal, less fateful than those incidents that lead towards the end of a
tragedy in the poets? and this was a tragedy. Oh, how sad, how pitiful, to
the Joyce of twenty years ago! but not to our Joyce, who suddenly found
this July morning her vague dreams of youth, her fancies that had no
foundation, coming true.
‘You’ve been a long time away,’ said Janet from the door. She had
watched Joyce’s approach until they were within a few steps of each other,
when she had suddenly withdrawn her eyes, and taking to examining the
hem of her apron, which she laid down and pinched between her fingers, as
if preparing it to be hemmed over again. The corners of Janet’s mouth were
drawn down, and a line or two marked in her forehead, as when she was
angry and about to scold her nursling. ‘I could wuss,’ she said, ‘that ye
wouldna stravaig away in the mornin’ without a piece or onything to sustain
ye, and maybe getting your death o’ cauld, sittin’ on the grass.’
‘It is the first day of the holidays, granny,’ said Joyce. She came in
smiling, and put down her book, and going up to her faithful guardian, put
an arm round her, and laid her cheek against hers. Caresses are rare in a
Scotch peasant’s house. Janet half turned away her own wrinkled cheek.
The intensity of the love within her rose into a heat which simulated wrath.

‘I’m no a wean to be made o’. I like nane o’ your phrasin’s. I like when
folk do as I bid them, and make nae steer.’
‘Oh, granny,’ said Joyce, ‘but my heart is so full, and I have so much to
tell you.’
‘What can ye have to tell me? I have maybe mair to tell you than ever ye
thought upon; and as for a full heart, how can the like of you, with a’ your
life before ye, ken what that means?’
‘Granny, I have had a long talk with that gentleman—the gentleman that
thought he knew my mother.’
‘And what had he to say to you? I’m thinking your mother has been just
killed among them. That’s my opinion. A poor young solitary thing, that had
naebody to stand up for her. And sae will ye be if ye lippen to them,’ cried
Janet, suddenly sitting down and covering her face with her apron,—‘sae
will ye be. Ye are weel off now, though maybe ye dinna think sae.’
‘Granny, have I ever given you any reason to say that?’
Janet withdrew her apron from her eyes. Her eyes were red with that
burden of tears which age cannot shed like youth. The passion of love and
grief which overflowed her being could only get vent in this irritation and
querulous impatience. Her long upper lip quivered, a hot moisture glistened
on the edges of her eyelids. She looked at the young creature, standing half
on the defensive before this sudden attack, yet half disposed to meet it with
tender laughter and jest. ‘Oh, ye can make licht o’t,’ she cried. ‘What is’t to
you? just the life ye’ve aye been craving for,—aye craving for,—ye canna
say nay. But to me what is it?’ said the old woman. ‘It’s just death. It’s waur
than death; it’s just lingering and longin’ and frettin’ wi’ my Maker for what
I canna have! When we took ye to our airms, a bit helpless bairn, maybe
there was that in our hearts that said the Lord was our debtor to make it up
to us. But them that think sae will find themselves sair mista’en; for He has
just waited and waited till ye had come to your flower and were our pride!
And now the fiat has gaen forth, no’ when ye were a little bairn; and I aye
said, “Haud a loose grip!” But now that a’ the danger seemed overpast, now
that—wheesht!’ cried Janet suddenly, coming to an abrupt pause. In the
silence that followed they heard a slow and heavy foot, making long and
measured steps, advancing gradually. They heard that among many others,
for it was the time when the labourers were coming home to dinner; but to
Janet and Joyce there was no mistaking the one tread among so many. Janet

got up hurriedly from the chair. ‘Wheesht! no’ a word before him; it’s time
enough when it comes,’ she said. Joyce had not waited even for this, but
had begun to lay the table, so that Peter when he came in should find
everything ready. He came in with his usual air of broadly smiling
expectation, and took his bonnet from his grizzled red locks, which was the
fashion Joyce had taught him, as he stepped across the threshold. ‘It’s awful
warm the day,’ were his first words, as he went in, notwithstanding, and
placed himself in the big chair near the fire. The fire was the household
centre whether it was cold or warm. ‘So you’ve gotten the play?’ he added,
beaming upon Joyce, awaiting something which should make him open his
mouth in one of those big brief laughs that brought the water to his eyes. It
was not necessary that it should be witty or clever. Joyce was wit and
cleverness embodied to her foster-father. When she opened her lips his soul
was satisfied.
And before Peter the cloud disappeared like magic. Janet was cheerful,
and Joyce like everyday. They listened to his talk about the ripening corn,
and where it was full in the ear, and where stubby, and about the Irish
shearers that will be doun upon us like locusts afore we ken,—‘and a wheen
Hieland cattle too,’ said Peter, who was not favourable to the Celts. Then
the broth was put on the table and the blessing said, and the humble dinner
eaten as it had been for years in the little family which held together by
nature, and which, so far as had appeared, nothing could ever divide.

CHAPTER IX
The Colonel took his wife’s arm, drawing her close to him, leaning over her
little figure: he could hold her closer in this way, and take her strength more
completely into his own than if she had taken his arm in the ordinary
fashion. But she gave him but an uncertain support for the first time in their
life. The group made up of those two figures linked into one, making but
one shadow, tottered as they set out. And she made no reply to his look, to
the urgent clasp of his arm on hers, until they had passed out of the village
street, and gained the quiet and stillness of the avenue within the gates.
Then Elizabeth—unprecedented action!—detached herself almost with
impatience. ‘You hurt me, Henry,’ she said quickly, with a sharp intolerance
in her tone. This brought the painful excitement of the morning to a climax;
for when had she complained before?
‘My dear!’ he cried, with a tone of compunction and horror, ‘I—hurt
you?’ as if he had been accused of high treason and brutal cruelty
combined.
This accent of amazed contrition brought Mrs. Hayward to herself. ‘Oh
no, Henry,’ she said, ‘you did not hurt me at all. I am not fit to speak to any
good Christian. I am a wretched creature, full of envy, and malice, and all
uncharitableness. Let me alone a little till I come to myself.’
The Colonel gave her a piteous look. ‘As long as you please, my dear,’
he said; then added apologetically, ‘I can’t help feeling very anxious. There
is more in this than meets the eye—there is more in it than I realised: there
is—the—the young lady, Elizabeth.’
In spite of herself his wife looked at him with a momentary scorn which
was almost fierce. ‘Do you mean to say that this is the first time you have
thought of that?’
The Colonel was very apologetic. ‘I am afraid I am dense,’ he said; ‘but,
my dear, I always like to wait till I know what you think—and as yet you
have said nothing. How was I to suppose——’ Here he broke off, seeing in
his wife’s eyes more than he could read all at once, and with a tremulous
movement laid his hand again upon her arm. ‘What is it?’ he said.
She was tremulous too, but in a different fashion. She began to open out
a little parcel which she held in her hand quickly, almost with indignation.

‘You will know what to think when you see you own hand and name,’ she
said. ‘There! that’s been laid up waiting for me—fancy! for me to find it—
these twenty years.’
The Colonel looked at the yellow old letters with increasing agitation,
but no increase of understanding. ‘What is it?’ he said. ‘What does it mean,
Elizabeth? I did not go through all this, only to come to an old letter of my
own at the last.’
The little woman stamped her foot with a kind of fury. ‘I think you are
determined not to understand,’ she cried. ‘Look who that letter is addressed
to—look at this other along with it; for God’s sake, Henry, don’t worry me
any more! don’t ask what I think: look at them for yourself.’
He did look, but with so bewildered an expression that compassion
overcame her. She took the papers over which he was puzzling, looking at
his own writing vaguely, with a quick impatient movement.
‘You have been right, quite right in your conjectures,’ she said; ‘the poor
girl that came here alone twenty years ago, and had her baby, and went
wrong in her head, and died, was your poor young wife, Joyce Hayward,
Henry. There is your letter to her—not the kind of letter I should have
thought you would have written; and there is hers to you, a voice out of the
grave. Don’t look at me in that pitiful way. I don’t expect you to read it
here. Go away to your own room or into the woods, Henry, and read your
wife’s letter. Go away! go away! and do this for yourself without me. I am
not the person,’ cried Mrs. Hayward, thrusting them into his hands, and
pushing him impatiently from her,— ‘I am not the person to read your
wife’s letter. Go away! go away!’
‘My wife’s letter,’ he said, with a momentary look of awe and trouble.
Then suddenly he put one arm round her, and, half sobbing, said, ‘Twenty
years since! it has always been right, all the time, my darling, between you
and me.’
‘Oh, Henry!—is that all you think of at such a moment?’
He patted her shoulder with his large and unsteady hand, and held her
close. ‘If it is not all, it’s the first and foremost,’ he said; ‘you will never
again, Elizabeth, never any more——’
‘Oh, go away! go away!’ she cried, stamping her foot upon the path.
There were tears in her eyes, half love and softness, half impatience and
fury. She pushed him away from her with all her strength, and turning her

back upon him, walked quickly through the trees and across the park in the
full sunshine. She was distracted with conflicting sentiments, unwilling to
be melted, yet touched to the heart; determined that he should go back by
himself into that distant past with which she had nothing to do, yet scarcely
able to resist the habit of doing everything for him, of encountering even
that for him. She hurried along until she had got within the shade of a belt
of wood, and out of sight of the spot where she had left her husband. Here
Mrs. Hayward suddenly sat down upon the grass, and hid her face in her
hands. Sometimes it became necessary for her, even in the ordinary course
of affairs, to escape for a moment now and then from the Colonel’s constant
demands. But to-day it seemed to her that she must do this or die. The
sudden summons, the long journey, the agitating news, the commission so
suddenly put into her hands, the discovery she had made, all united had
overwhelmed her at last. She cried heartily, as she did everything, with an
abundant natural overthrow of feeling which relieved and exhausted her,
and a sensation underneath all which she could not define whether it was
happiness or pain.
This Joyce, who had been from the beginning the shadow upon her
married life, in despite of whose possible claims she had married, and
whom she had regarded all through with a mixture of pity and indignation
and fear, roused in her, dead, almost as strong feelings as if she had been a
living claimant to the name and place which were hers. The very fact that
the poor girl’s story was so pitiful, and that nothing could take away the
interest and compassion roused by the image of a young forsaken creature
dying so miserably with no one near who loved her, was to Mrs. Hayward
at this moment an additional aggravation, adding a pang to all the rest. And
yet there was in it an unspeakable relief; and the fact that this, and not any
revival of the romance of his youth, had been her husband’s first thought,
was exquisite to her, yet with a certain acrid sweetness, not unmingled with
pain and the contradictoriness of a highly sensitive, impatient, and
intolerant soul, sharply conscious of every complication. For
notwithstanding her strong personal share in the matter, it was clear to
Elizabeth that he ought to have thought of the other, the poor girl in her
youth and misery, first; and that the sight of her letter, the words written in
her anguish, coming to him as it were from her grave, across the silence of
twenty years, ought to have transported the man to whom these words were
addressed out of all recollection of the present,—out of everything save that

tragedy of which, however innocently, he was the cause. She could not but
feel it sweet that it was herself and not the dead Joyce of whom in reality he
had thought: yet, in a manner, she resented it, and was wounded by it as a
thing against nature which ought not to have been. ‘That is all that a man’s
love is worth,’ she said to herself. ‘He cost her her life, and it is me he
thinks of, who am well and strong, and in no trouble.’ And yet it went to her
heart that he should have so thought.
In this keen complication of feeling, Mrs. Hayward, for the time, could
realise nothing else. It was not possible to think of the dead girl and herself
but as rivals: and this, too, gave her a pang. How mean, how ungenerous,
how miserable it was! Such a story in a book, much more in real life, would
have moved her to warm tears; but in this, which touched herself so closely,
she could feel no true pity. It was her rival; it was one who had come before
her, whose shadow had lain upon her life and darkened it, who even now
was bringing trouble into it—trouble of which it was impossible to fathom
the full extent. How could there be tenderness where such sharp antagonism
was? And yet, how poor, how small, how petty, how unworthy was the
feeling!
In these contrarieties her mind was caught, and thrilled with sharp
vexation, shame, scorn of herself, and sense of that profound vanity of
human things which makes the present in its pettiness so much greater than
the past, and dims and obliterates everything that is over. To think that such
a tragedy had been, and that those who were most concerned thought of
their poor share in it first, and not of her who was the victim! That
contradiction of all that was most true and just, that infidelity which is in
every human thing, the callousness and egotism which ran through the best,
jarred her with a discord which was in herself as well as in all the rest. But
when she had cried her heart out, Mrs. Hayward, as was natural, exhausted
that first poignant sensation, and came to contemplate, apart from all that
was past, the present condition of affairs, which was not more consolatory.
Indeed, when, putting the tragedy of the poor Joyce who was dead out of
her mind, she returned to the present, the figure of the living Joyce suddenly
rose before her with a sharp distinctness that made her spring to her feet as
a soldier springs to his weapon when suddenly confronted by an enemy.
Mrs. Hayward had never seen Joyce, so that this figure was purely
imaginary which rose before her, with a stinging touch, reminding her that
here was something which was not past but present, a reality,—no affair of

memory or sentiment, but a difficulty real and tangible, standing straight
before her, not to be passed by or forgotten. She sprang up as if to arms, to
meet the new antagonist who thus presented herself, and must be met, but
not with arms in hand, nor as an antagonist at all. Joyce herself would
scarcely have been so terrible to encounter as Joyce’s child thus coming
between her husband and herself, taking possession of the foreground of
their existence whether they would or not. What Mrs. Hayward would be
called upon to do would be—not to retire before this new actor in her
existence, not to withdraw and leave the field as she had always felt it
possible she might have to do, but to receive, to live with,—good heavens!
perhaps to love her! Yes! no doubt this was what the Colonel would want;
he would require her to love this girl who was his child. He would take it
for granted that she must do so; he would innocently lay all the burden upon
her, and force her into a maternity which nature had not required of her. A
mother! ah yes, she could have been a mother indeed had God willed it so;
but to produce that undeveloped side of her, that capacity which she had
been so often tempted to think Providence had wronged her by leaving in
abeyance, for the benefit of this country girl, this Scotch peasant, with all
her crude education, her conceit (no doubt) of superiority, her odious
schoolmistress’s training!
Mrs. Hayward could not sit still and look calmly at what was before her.
There was something intolerable in it, which stung her into energy, which
made her feel the necessity of being up and doing, of making a stand
against misfortune. However much she might resent and resist in her private
soul, she would have to do this thing, and put on a semblance of doing it
with, not against, her own will and liking. Talk of the contradictions of fate!
they seemed to be all grouped together in this problem which she had to
work out. If the child had been a boy, the Colonel would have been
compelled more or less to take the charge upon himself. There would have
been school or college, or the necessities of a profession, to occupy the
newcomer; but that it should be a girl—a girl, a young woman, a creature
entirely within the sphere of Colonel Hayward’s wife, whose business it
would be not only to be a mother to her, but to receive her as a companion,
to amend her manners, to watch over all her proceedings, to take the
responsibility night and day!
Mrs. Hayward felt that she could have put up with a boy. He would not
have been her business so much as his father’s, and he would not for ever

and ever have recalled his mother, and put her in mind of all that had been,
and of all she herself had already borne. For though she had accepted the
position knowing all that was involved, and though it was, so to speak, her
own fault that she had encountered these difficulties, still there could be no
doubt that she had for years had much to bear; and now what a climax, what
a crown to everything! A second Joyce, no doubt, with all the headstrong
qualities which had made the first Joyce spoil her own life and the lives of
others, with all the disadvantages of her peasant training, of her education
even, which would be rather worse than ignorance. Mrs. Hayward conjured
up before her the image of a pupil-teacher, a good girl striving for
examinations, immaculate in spelling, thinking of everything as the subject
of a lesson: looking up with awe to the inspector, with reverence to some
little prig of a schoolmaster, a girl with neat collars and cuffs, knowing her
own condition in life, and very respectful to her superiors: or else
bumptious, and standing upon her dignity as an educated person, which
Mrs. Hayward had heard was more the way of the Scotch. In either point of
view, what a prospect, what a companion!
And the Colonel’s wife knew how that good man would conduct
himself. He would remonstrate with her if the girl were gauche, or if she
were disagreeable and presuming. He would say, ‘You must tell her’—‘you
must make her do so-and-so.’ If his taste was shocked, if the girl turned out
to be very dreadful, he himself, who ought to know so much better, would
throw all the blame upon her. Or perhaps, which would be still more
intolerable, his eyes would be blinded, and he would see nothing that was
not beautiful and amiable in his child. With a sudden flush of irritation,
Mrs. Hayward felt that this would be more unbearable still. Joyce had been
the bugbear of his life in the past; what if Joyce were to be the model, the
example of every good quality, the admiration and delight of his life to
come: and she herself, the step-mother, the half-rival, half-tyrant, the one
who would not appreciate the new heroine! No one was so ready as
Elizabeth to perceive all her husband’s excellent qualities. He was good as
an angel or a child—there was no soil in him. His kindness, his tenderness,
his generous heart, his innocent life, were her pride and delight. And the
perpetual appeal which he made to her, the helplessness with which he
flung himself upon her for inspiration and counsel, made him dearer still.
She herself laughed and sometimes frowned at the devout aspiration, ‘If
only Elizabeth were here!’ for which all his friends smiled at the Colonel;

but at the same time it warmed her heart. And yet there was no one in the
world so feelingly alive to the irritations and vexations which were involved
in this supreme helplessness and trust. There were moments when he
worried her almost beyond endurance. She had to be perpetually on the
watch. She had to subdue herself and forget herself, and make a thousand
daily sacrifices to the man whom she ruled absolutely, and who was ready
at her fiat almost to live or die. But of all intolerable things, that which was
most intolerable was the suggestion that he might in this matter judge for
himself without her aid,—that he might admit this strange girl into his
heart, and place her on the pinnacle which had hitherto been sacred to
Elizabeth alone.
She had seated herself on a grassy bank under the shade of the trees
which skirted one side of the park of Bellendean. Instinctively she had
chosen a spot where there was ‘a view.’ How many such spots are there to
which preoccupied people, with something to think out, resort half
unawares, and all-unconscious of the landscape spread before them!
Edinburgh, gray in the distance, with her crags and towers, shone through
the opening carefully cut in the trees, the angle of the castled rock standing
forth boldly against the dimness of the smoke behind; and the air was so
clear, and the atmosphere so still, that while Mrs. Hayward sat there the
sound of the gun which regulates the time for all Edinburgh—the gun fired
from the Castle at one o’clock—boomed through the distance with a sudden
shock which made her start. She was not a fanciful woman, nor given to
metaphors. But there was something in the peace of the landscape, the
summer quiet, broken only by the hum of insects and rustle of the waving
boughs, the distant town too far off to add a note to that soft breathing of
nature, which made a centre to the picture and no more—when the air was
suddenly rent by the harsh and fatal sound of the gun, making the spectator
start—which was to her like an emblematic representation of what had
happened to herself. To be sure, if she had but thought of it, that voice of
war had been tamed into a service of domestic peace, a sound as innocent as
chanticleer; but Mrs. Hayward was a stranger, and was unaware of this. As
she rose up hurriedly, startled by the shock in the air, she saw her husband
coming towards her across the sunshine. He was moving like a man in a
dream, moving instinctively towards where she was, but otherwise
unconscious where he was going, unaware of the little heights and hollows,
stumbling over the stump of a tree that came in his way. The sight of his

abstraction brought her back to herself. He came up to her, and held out the
little packet in his hand.
‘Put them away,’ he said hoarsely; ‘lock them up in some sure place,
Elizabeth. To think all that should have been going on, and I ignorant—oh,
as ignorant as the babe unborn!’
‘How could you know when she never told you?’ Mrs. Hayward cried
quickly, instinctively taking his part, even against himself. He put his large
hand upon her small shoulder, and patted her with a deprecating, soothing
touch, as if the wrong and the sorrow were not his but hers.
‘But she meant us to know—that letter, if I had ever got it! She was
young and foolish, young and foolish. Put it away, my dear; don’t destroy it,
but lock it away safe, and let us think of it no more.’
‘That is impossible, Henry. You must think of it, in justice to her—poor
thing;’ this Mrs. Hayward said unwillingly, from a sense of what was right
and fitting, and with a compunction in her heart,—‘and for the sake,’ she
added firmly, after a moment, ‘of your child.’
‘The girl,’ he said vaguely. Then he came closer to her, and put his arm
within hers. ‘You will see to all that, Elizabeth. You understand these sort of
things better than I do. It would be very awkward for me, you know, a
man.’ To describe the persuasive tone, the ingratiating gesture with which,
in his simplicity, he put this burden upon her, would be impossible. Even
she, well as she knew him, was struck with surprise—a surprise which was
half happiness and half indignation.
‘Henry!’ she cried, resisting the appealing touch, ‘have you no heart for
your own child?’
He leant upon her for a moment, drawing as it seemed her whole little
person, and all her energy and strength, into himself. ‘I’m all upset,
Elizabeth. I don’t know what I have, whether heart or anything else—
except you, my dear, except you. Everything will go right as long as I have
you.’

CHAPTER X
In the perplexity of this extraordinary crisis they both went, without another
word, ‘home’: though it was no more home than these wonderful new
circumstances were the course of everyday. If we were to prophesy the
conduct of human creatures in moments of great emotion by what would
seem probable, or even natural, how far from the fact we should be!
Colonel Hayward, a man of the tenderest heart and warmest affections,
suddenly discovers that he has a child—a child by whose appearance, and
everything about her, he has been pleased and attracted, the child of his first
love, his young wife to whose cruel death he has contributed, though
unwittingly, unintentionally, meaning no evil. Would not all ordinary means
of conveyance be too slow, all obstacles as nothing in his way, the very
movement of the world arrested till he had taken this abandoned child into
his arms, and assured her of his penitence, his joy, his love! But nothing
could be further from his actual action. He went back to Bellendean with a
feeling that he would perhaps know better what to do were he within the
four walls of a room where he could shut himself and be alone. It would be
easier to think there than in the park, where everything was in perpetual
motion, leaves rustling, branches waving, birds singing,—the whole world
astir. ‘If we were only in our own room,’ he said to his wife, ‘we could
think—what it was best to do.’
She said nothing, but she longed also for the quiet and shelter of that
room. She recognised, as indeed she might have done from the first, that
whatever had to be done, it was she that must do it. And Mrs. Hayward was
entirely dépaysée, and did not know how to manage this business. Janet
Matheson was a new species to a woman who had done a great deal of
parish work, and was not unacquainted with the ordinary ways of managing
‘the poor.’ She did not understand how to deal with that proud old woman,
to whom she could not offer any recompense, whom she would scarcely
dare even to thank for her ‘kindness.’ Janet had repudiated that injurious
word, and Mrs. Hayward felt that it would be easier to offer money to Mrs.
Bellendean than to this extraordinary cottager. To be sure, that was nothing
—a trifle not worth consideration in face of the other question, of Joyce
herself, who would have to be adopted, removed from the cottage, taken

home as Miss Hayward, a new, and perhaps soon the most important,
member of the family. Elizabeth’s heart beat as it had never done before,
scarcely even when she married Captain Hayward, accepting all the risks,
taking him and his incoherent story at a terrible venture. That was an
undertaking grave enough, but this was more terrible still. She felt, too, that
she would be thankful to get into the quiet of her own room to think it over,
to decide what she should best do.
This, however, was more easily said than done. The anxious pair were
met in the hall by Mrs. Bellendean with looks as anxious as their own. She
was breathless with interest, expectation, and excitement: and came up to
them in a fever of eagerness, which, to Mrs. Hayward at least, seemed quite
unnecessary, holding out a hand to each. ‘Well?’ she cried, as if their secrets
were hers, and her interest as legitimate as their own. In short, the pair, who
were very grave and preoccupied, having exhausted the first passion of the
discovery, had much less appearance of excitement and expectation than
this lady, who had nothing whatever to do with it. A shade of
disappointment crossed her face when she saw their grave looks; but Mrs.
Bellendean’s perceptions were lively, and she perceived at the same
moment tokens of agitation in the old colonel’s face which reassured her. It
would have been too much if, after all her highly-raised expectations,
nothing had happened at all.
‘Come into my room,’ she said quickly; ‘we have half an hour before
luncheon, and there we shall be quite undisturbed.’ She led the way with a
rapidity that made it impossible even to protest, and opening the door,
swept them in before her, and drew an easy-chair forward for Mrs.
Hayward. ‘Now,’ she said, ‘tell me! You have found out something, I can
see.’
They looked at each other,—Mrs. Hayward with the liveliest inclination
to tell the lady, whom she scarcely knew, that their affairs were their own. It
would have been a little relief to her feelings could she have done so; but
this was just the moment, as she knew very well, in which the Colonel was
sure to come to the front.
‘Yes,’ he said, with a sigh, in which there was distinct relief. (He found it
so easy to relieve himself in that way!) ‘We have found out—all we wanted,
more than we expected. Apart from all other circumstances, this is a

memorable visit to me, Mrs. Bellendean. We have found—or rather
Elizabeth has found—— She is always my resource in everything——’
‘What?’ cried Mrs. Bellendean, clasping her hands. ‘Please excuse me—
I am so anxious. Something about Joyce?’
‘You must understand that I had no notion of it, no idea of it all the time.
I was as ignorant—— There may have been things in which I was to blame
—though never with any meaning: but of this I had no idea—none: she
never gave me the slightest hint—never the least,’ said the Colonel
earnestly. ‘How could I imagine for a moment—when she never said a
word?’
Mrs. Bellendean looked at Mrs. Hayward with an appeal for help, but
she gave a smile and glance of sympathy to the Colonel, who seemed to
want them most. His wife sat very straight, with her shoulders square, and
her feet just visible beneath her gown—very firm little feet, set down
steadily, one of them beating a faint tattoo of impatience on the carpet. She
was all resistance, intending, it was apparent, to reveal as little as possible;
but the Colonel, though his style was involved, was most willing to explain.
‘It is,’ he said, ‘my dear lady, I assure you, as much a wonder and
revelation to me as to any one. I never thought of such a possibility—never.
Elizabeth knows that nothing was further from my mind.’
‘Henry,’ said his wife suddenly, ‘you have been very much agitated this
morning. All these old stories coming up again have given you a shake. Go
up, my dear, to your room, and I will tell Mrs. Bellendean all that she cares
to hear.’
‘Eh? do you think so, Elizabeth? I have got a shake. It agitates a man
very much to be carried back twenty years. Perhaps you are right: you can
explain everything—much better than I can—much better always; and if
Mrs. Bellendean thinks I am to blame, she need not be embarrassed about
it, as she might be before me. I think you are right, as you always are. And
perhaps she will give you some good advice, my love, as to what we ought
to do.’
‘I am sure I shall not think you to blame, Colonel Hayward,’ cried Mrs.
Bellendean, with that impulse of general amiability which completed the
exasperation with which Elizabeth sat looking on.
‘Yes, no doubt, she will give me good advice,’ she said, with
irrepressible irritation; ‘oh, no doubt, no doubt!—most people do. Henry,

take mine for the moment, and go upstairs and rest a little. Remember you
have to meet all the gentlemen at luncheon: and after that there will be a
great deal to do.’
‘I think I will, my dear,’ Colonel Hayward said: but he paused again at
the door with renewed apologies and doubts—‘if Mrs. Bellendean will not
think it rude, and even cowardly, of me, Elizabeth, to leave all the
explanations to you.’
Finally, when Mrs. Bellendean had assured him that she would not do so,
he withdrew slowly, not half sure that, after all, he ought not to return and
take the task of the explanation into his own hands. There was not a word
said between the ladies until the sound of his steps, a little hesitating at first,
as if he had half a mind to come back, had grown firmer, and at last died
away. Then Mrs. Hayward for the first time looked at the mistress of the
house, who, half amused, half annoyed, and full of anxiety and expectation,
had been looking at her, as keenly as politeness permitted, from every point
of view.
‘My husband has been very much agitated—you will not wonder when I
tell you all; and he is never very good at telling his own story. A man who
can do—what he can do—may be excused if he is a little deficient in
words.’
She spoke quickly, almost sharply, with a little air of defiance, yet with
moisture in her eyes.
‘Surely,’ said Mrs. Bellendean, ‘we know what Colonel Hayward is; but
pardon me, it was a much less matter—it was about Joyce I wanted to
know.’
‘The one story cannot be told without the other. My husband,’ said Mrs.
Hayward, with a long breath, ‘had been married before—before he married
me. He had married very hurriedly a young lady who came out to some
distant relations in India. They were at a small station out of the way. She
was not happy, and he married her in a great hurry. Afterwards, when she
was in England by herself, having come home for her health, some wicked
person put it into the poor thing’s head that her marriage was not a good
one. She was fool enough to believe it, though she knew Henry. Forgive me
if I speak a little hastily. She ought to have known better, knowing him; but
some people never know you, though you live by their side a hundred
years.’

She stopped to exhale another long breath of excitement and agitation. It
was cruel to impute blame to the poor dead girl, and she felt this, but could
not refrain.
‘And suddenly, after one letter full of complaint and reproach, she wrote
no more. He was in active service, and could not get home. It was not so
easy then to come home on leave. He wrote again and again, and when he
got no answer, employed people to find her out. I can’t tell you all the
things that were done—everything, so far as he knew how to do it. I didn’t
know him then. I daresay he wasted a great deal of money without getting
hold of the right people. He never heard anything more of her, never a
word, till the other day.’
‘Then that poor young creature was—— And Joyce—Joyce!—who is
Joyce? Mrs. Hayward, do you mean really that Joyce——’
‘Joyce—was his first wife: and this girl—who has the same name,—I
have not seen her, I don’t know her, I can express no feeling about her,—
this young lady is my husband’s daughter, Mrs. Bellendean.’
‘Colonel Hayward’s daughter!’ Mrs. Bellendean sprang to her feet in her
surprise and excitement. She threw up her hands in wonder and delight and
sympathy, her eyes glittered and shone, a flush of feeling came over her.
Any spectator who had seen the two ladies at this moment would have
concluded naturally that it was Mrs. Bellendean who was the person chiefly
concerned, while the little woman seated opposite to her was a somewhat
cynical looker-on, to whom it was apparent that the warmth of feeling thus
displayed was not quite genuine. The Colonel’s wife was moved by no
enthusiasm. She sat rigid, motionless, except for that one foot, which
continued to beat upon the carpet a little impatient measure of its own.
‘Oh,’ cried Mrs. Bellendean, ‘I always knew it! One may deceive one’s
self about many people, but there was no possibility with Joyce. She was—
she is—I never saw any one like her—quite, quite unprecedented in such a
place as this: like nobody about her—a girl whom any one might be proud
of—a girl who—oh yes, yes! you are right in calling her a young lady. She
could be nothing less. I always knew it was so.’
‘She is my husband’s daughter,’ said Mrs. Hayward, without moving a
muscle. She remained unaffected by her companion’s enthusiasm. She
recognised it as part of the burden laid upon her that she should have to
receive the outflowings of a rapture in which she had no share.

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Neurotransmitter Transporters Structure Function and Regulation 2nd Edition Sammanda Ramamoorthy (Auth.)

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