Events at the neuromuscular junction.

1st year medical school physiology essay:
Describe the effects between the action potential arriving at the axon terminal and skeletal muscle contraction.

1. Christiane – HOM3 1
Describe the effects between the action potential arriving at the axon terminal
and skeletal muscle contraction.

I am just talking about skeletal muscle here.

Brief summary of events
Each event described is causal to the next one, unless otherwise stated:
- An action potential (AP) is propagated along the length of the axon
- Voltage gated calcium channels open and allow influx of calcium into the
terminal bouton
- Vesicles filled with the neurotransmitter acetylcholine move to the surface and
release acetylcholine into the synaptic cleft
- Acetylcholine diffuses across the cleft and binds its receptor (2 ACh per
receptor)
- the acetylcholine receptor (a Na+/K+ channel) opens and renders the
membrane permeable to Na+/K+ ions
- the ion flux causes depolarisation which is passed onto the edges of the motor
end plate
- an action potential occurs in the membrane of the muscle cell adjacent to the
motor end plate
- the action potential is propagated along the membrane and enters the T
tubules, which lead vertically into the muscle fibre
- voltage sensing dihydropyridine receptors in the T tubular membrane activate
ryanodine receptors in the sarcoplasmic reticulum, resulting in a Ca2+ influx
into the sarcoplasm, where the contraction machinery is located
- Ca2+ reaches the myofibrils and binds to the protein troponin, releasing
tropomysin from the myosin-binding site of actin
- Actin binds myosin, an ATPase pre-charged with ATP which binds actin in its
high-energy state
- Myosin releases ADP/Pi and returns to the low-energy state, thereby pulling
the actin filaments closer towards the M-line
- The sarcomere shortens
- (not causal) another ATP binds to myosin
- myosin dissociates from actin and becomes re-activated as it hydrolyses ATP
to ADP + Pi
- activated myosin re-binds to the actin filament and causes further shortening
of the sarcomere
- (not causal) if actin-myosin binding is repeated often enough and in a number
of myofibrils, the muscle contracts
- the end: as the initial stimulus comes to an end, the release of Ca2+ into the
sarcoplasm ceases, and Ca2+ is pumped back into the sarcoplasmic reticulum
by a Ca2+ pump (this pump is always active). Tropomysin re-binds to actin
and myosin can no longer bind actin, preventing further contraction.

2. Christiane – HOM3 2
IN DETAIL
Events at the neuromuscular junction
http://www.anaesthesiauk.com/images/nmj1.jpg

- the action potential travels down the length of the axon and arrives at the
neuromuscular junction
- the end of the axon is a knoblike structure called the terminal bouton, and fits
into a wavy depression on the muscle fibre, called the motor end plate
- the terminal bouton is rich in voltage-gated Ca2+ channels (brown lines),
which open in response to the membrane depolarisation caused by the arriving
action potential
- as a result, calcium ions move into the terminal button, which in turn causes
vesicles filled with the neurotransmitter acetylcholine (ACh) to surface,
merge with the membrane, and release ACh into the synaptic cleft
- ACh diffuses across the synaptic cleft (contributes a lot to overall transmission
time since speed of diffusion is limited), where it binds to ACh receptors in
the motor end plate membrane (nicotinic type receptors)
- ACh receptors are ligand-gated Na+/K+ channels
- open in response to ACh binding and let Na+/K+ pass through the membrane
(The cellular concentration of Na+ is high outside and low inside, the
concentration of K+ is low outside and high inside. The intracellular matrix is
more negative compared to the inside. As a result, more Na+ ions move in
than K+ ions move out)
Terminal button
Motor end plate
Muscle cell!
Figure 1

3. Christiane – HOM3 3
- The migration of Na+/K+ causes membrane depolarisation (end plate
potential, EPP, a graded potential)
- Since the motor end plate is located in the middle of the muscle cell, the EPP
is carried to the edges of the motor end plate in two directions, where it causes
an action potential in the muscle fibre membrane
- Note: for each AP in the terminal button, APs occur in the muscle fibre. In
synapses (i.e. junctions between two nerve cells), the AP at the terminal button
brings about a post-synaptic potential that is either excitatory or inhibitory,
and the summation of these will determine whether a post-synaptic action
potential occurs or not
- The enzyme acetylcholine esterase is present in the folds of the motor end
plate membrane (extracellularly). It decomposes ACh after it has dissociated
from its receptor (binding is continuous process of association and
dissociation and as more ACh arrives at the receptor site, it moves down into
the folds of the motor endplate membrane, where it is degraded).

Structure of a muscle fibre
To understand how the action potential generated at the neuromuscular junction
causes muscle contraction, it is useful to understand the structure of a muscle cell:
http://people.eku.edu/ritchisong/301images/Muscle_cell_Nature.jpg
Figure 2

4. Christiane – HOM3 4
- Muscle fibres are elongated multinucleated cells containing many
mitochondria and many many myofibrils (~80% of the volume of the muscle
cell), responsible for the contractile function of the muscles
- a single muscle cell can be up to 30cm in length and has a diameter of 10-
100um
- because of their typical appearance and structure, particular parts of a muscle
cell have been renamed, for example the membrane of the muscle cell is called
“sarcolemma”, the endoplasmatic reticulum “sarcoplasmatic reticulum”, the
cytoplasm “sarcoplasm”… (greek: sarx = flesh, meros = part)
- myofibrils consist of the “contracting” proteins actin and myosin (even though
the proteins themselves don’t contract) and regulatory proteins such as
tropoponin and tropomysin (Figure 3).
- Actin is a 42kDa globular protein which assembles into two-stranded helical
filaments in an ATP dependent process (like “beads on a string”)
- Myosin is a much larger multi sub-unit multi domain protein complex, with an
overall “golf-club-like” appearance. It consists of a tail, a neck and a head
domain. The head domain contains the actin and an ATP binding-site, the
head therefore has ATPase activity
- Tropomyosin is an alpha-helical coiled coil protein binding to the actin
filament along its length, covering up the Myosin binding-site in the resting
state.
- Troponin is a trimeric protein consisting of TnC, TnT and TnI subunits. The
TnT subunit is in contact with tropomyosin.
http://www.sigmaaldrich.com/etc/medialib/life-science/metabolomics/enzyme-
explorer/myosin.Par.0001.Image.490.gif
- The thin Actin filaments (blue lines in Figure 2) are held in place by a
cytoskeletal protein generating a double-comb-like structure (what is this
protein called?), into which the thick myosin filaments (red lines in Figure 2)
insert (the head of the golf-club is pointing towards the bottom of the comb)
- The bottom of the comb/i.e. the part where the actin filaments are connected is
called the Z-line. The area on either side of the Z-line (where there are only
actin filaments) until the start of the myosin-filaments is called the I-band
- The area where actin and myosin filaments overlap is called the A-band
- In the centre of the A-band, there are only myosin filaments, but no actin
filaments, the H-zone. In the middle of the H-zone (in mammalian cells) lies
the M-line.
Figure 3

5. Christiane – HOM3 5
- The area in-between two Z-lines is called sarcomere and is the smallest
contracting unit of a myofibril.
- As several myofibrils line up perfectly parallel to each other, muscle cells
appear “striated” under the microscope (at high enough magnification), with
dark A-bands alternating with lighter I-bands, perpendicular to the length of
the muscle cell. At high enough magnification, the Z-line becomes obvious as
a dark line in the middle of the I-band. M-lines are visible as two light lines in
the middle of the A-band.
- In the transverse section of a myofibril, one can see that six actin filaments
surround one myosin filament, and that the Myosin head has six “cross-
bridges” pointing into the direction of the actin filament

Events in the muscle fibre leading to muscle contraction
- The surface membrane of the muscle fibre propagating the action potential
inserts continuously deep into the muscle fibre, in the form of a transverse
tubule (T tubule)
- T tubules in mammals are located at the junctions between I and A bands
- Between each T tubule, a myofibril is enveloped by the sarcoplasmic
reticulum, a structure of tubes and sacs surrounding it like a mesh, with
expanded edges towards the T tubules (lateral sacs/terminal cistern).
- In skeletal muscle, a T-tubule is immediately adjacent to two lateral sacs of
the sarcoplasmic reticulum (although they don’t touch directly). This is called
a triad.
- The propagating action potential in the T-tubule causes the release of Ca2+ in
the lateral sacs of the sarcoplasmic reticulum:
o The membrane of the T tubule contains dihydropyridine receptors
which sense the voltage change caused by the incoming action
potential
o It opens to allow Ca2+ entry
o More importantly, it activates (via mechanical linkage, but no direct
interaction?) the ryanodine receptor located in the sarcoplasmic
reticulum
o This results in calcium influx from the lateral sacs of the
sarcoplasmatic reticulum into the sarcoplasm
- Ca2+ reaches the myofibrils and binds to troponin, inducing conformational
changes in its three subunits
- This pulls tropomyosin deeper into the groove revealing the Myosin binding-
site on actin.
- As actin and myosin bind (via the crossbridges on the myosin head), the
myosin head bends (Figure 4), applying tension to the actin molecule
- This pulls the thin actin fibres towards the M-line, causing the sarcomere to
shorten (the thin filaments are pulled inwards so that the area in which they
overlap with the thick filaments is longer, the areas in which they don’t
overlap shorter, the filaments themselves to not become shorter)
- The bending of the myosin head is the cause for the so-called power-stroke
which causes the actin and myosin filaments to slide towards each-other,
shortening the length of the sarcomere

6. Christiane – HOM3 6
http://www.colorado.edu/intphys/Class/IPHY3430-200/image/12-9.jpg
- Energetically, this is achieved by converting the chemical energy of ATP into
mechanical energy (see Figure 4, although I don’t like the angles between the
myosin head and its tail, they are different to the textbook/lecture handouts):
o Prior to actin-myosin binding, the myosin head binds an ATP molecule
and hydrolyses it to ADP and Pi, causing the myosin head to adapt a
high-energy conformation
o In the presence of Ca2+, the activated myosin head binds actin
o Upon actin-binding ADP/Pi are released from myosin, the myosin
head returns to its low-energy state (while still being attached to actin),
thereby pulling on the actin filament
o In order for the myosin head to detach from actin, a new ATP
molecule has to bind (if no fresh ATP is provided, the muscle remains
in the rigor state. After death, there’s no ATP  rigor mortis)
o Hydrolysis of ATP creates a re-activated myosin head, ready to bind to
actin and carry out the power stroke as soon as calcium levels rise
again.
- A single power-stroke will not cause enough contraction in the muscle,
repeated cycles of cross-bridge binding and bending cause the contraction of
the entire muscle
- As soon as the action potentials in the T-tubules cease, Ca2+ release is
stopped and Ca2+ levels return to normal by the help of the Ca2+ ATPase
pump, moving Ca2+ from the cytoplasm into the lateral sacs
Figure 4