Cell membrane physiology and pharmacology

about structure and function of cell membrane and drugs modulating cell membrane.

1. CELL MEMBRANE PHYSIOLOGY & PHARMACOLOGY Dr Shahid Saache Dept . of Pharmacology BJ GMC, Pune Mentor- Dr Sujeet Divhare

2. Cell Membrane The cell membrane is a thin semi-permeable membrane that surrounds the cytoplasm of a cell, enclosing its contents.

3. Fungal cell membrane

4. Why cell membrane is important to study??

5. History 1895- Charles Ernest Overton- layers surrounding cells are ”lipoids” made of lipids and cholesterol 1925- Gorter and Grendel proposed lipid bilayer model of cell membrane 1935- Danielli and Davson earliest molecular model of biomembranes including proteins with lipids. 1958- Robertsons says two protein layers are adsorbed to lipid bilayer. All membrane have same composition. 1972- The Fluid Mosaic Model of Singer and Nicolson . 1984-The Mattress Model by Mouritsen and Bloom .

6. COMPOSITION OF CELL MEMBRANE Lipids Phosopholipids Sterols Proteins Integral Peripheral Carbohydrates Glycolipids Glycoproteins

7. Phospholipids Fatty acid Phosphate Fatty acid tails hydrophobic Phosphate group head hydrophilic Arranged as a bilayer

8. Phospholipid bilayer polar hydrophilic heads nonpolar hydrophobic tails polar hydrophilic heads

9. Lipid composition varies across the two leaflets of the same membrane Changes in distribution have biological consequences Platelet is able to play its role in clot formation only when phosphatidylserine moves to outer leaflet . Phosphatidylserine exposure also act as marker for programmed cell death

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11. Fluidity of membrane

12. Important for exocytosis and endocytosis For membrane biogenesis Factors altering fluidity Temperature ↑….. Fluidity Cholesterol content ↑….. Fluidity 16 Role of Fluidity of membrane

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14. 18 More than lipids… In 1972, S.J. Singer & G. Nicolson proposed that membrane proteins are inserted into the phospholipid bilayer It’s like a fluid… It’s like a mosaic… It’s the Fluid Mosaic Model !

15. Membrane is a collage of proteins & other molecules embedded in the fluid matrix of the lipid bilayer Extracellular fluid Cholesterol Cytoplasm Glycolipid Transmembrane proteins Filaments of cytoskeleton Peripheral protein Glycoprotein Phospholipids

16. Membrane proteins Classified depending on type of interaction with bilayer. Integral membrane proteins- pass through bilayer Peripheral membrane proteins- associate with bilayer by non-covalent interactions Lipid-anchored proteins.

17. Lipid-anchored membrane proteins Covalently linked to membrane by short oligosachharide linked to a molecule of GPI e mbedded on outer leaflet. Example – S crapie protein PrP c Some linked to inner leaflet like Ras and Src proteins have been implicated in transformation of normal cell to malignant cell.

18. Lipid-anchored membrane proteins One of protein is responsible for sleeping sickness. Protozoan parasite carried by tsetse flies survives in blood by virtue of dense cell surface coat made of a GPI anchored glycoprotein .( Eg - Transamidase complex) Several hundreds of glycoprotein variants to invade host immune system. Trypanosome brucie

19. Six major functions of membrane proteins Transport Enzymatic activity Signal transduction Cell-cell recognition Intercellular joining Attachment to the cytoskeleton ECM

20. Mechanical structure – maintain the physical integrity of cell and hold the cytoskeleton in place. Selective permeability – Gases, hydrophobic and small non polar molecules can easily pass through it. Transport – certain molecules pass through passively other need various transporters. Markers and signalling – some surface proteins act as cell marker and helps in cell signalling . functions of cell membrane

21. Movement across the Cell Membrane

22. Diffusion 2nd Law of Thermodynamics governs biological systems universe tends towards disorder (entropy) Diffusion movement from high  low concentration

23. Diffusion Move from HIGH to LOW concentration “passive transport” no energy needed diffusion osmosis movement of water

24. Diffusion across cell membrane Cell membrane is the boundary between inside & outside… separates cell from its environment IN food carbohydrates sugars, proteins amino acids lipids salts, O 2 , H 2 O OUT waste ammonia salts CO 2 H 2 O products IN OUT

25. Diffusion through phospholipid bilayer What molecules can get through directly? fats & other lipids inside cell outside cell lipid salt aa H 2 O sugar NH 3 What molecules can NOT get through directly? polar molecules H 2 O ions salts, ammonia large molecules starches, proteins

26. Factors affecting rate of diffusion Temperature - Higher temperature → diffuse faster Surface area - Larger surface area → diffuse faster Concentration gradient - Higher gradient → diffuse faster Size of particles - smaller particles → diffuse faster Diffusion medium - Solid → slowest Liquid → faster Gas → fastest

27. Channels through cell membrane Membrane becomes semi-permeable with protein channels specific channels allow specific material across cell membrane inside cell outside cell sugar aa H 2 O salt NH 3

28. Facilitated Diffusion Diffusion through protein channels channels move specific molecules across cell membrane no energy needed open channel = fast transport facilitated = with help high low

29. Active Transport conformational change Cells may need to move molecules against concentration gradient shape change transports solute from one side of membrane to other protein “pump” “costs” energy = ATP ATP low high

30. symport antiport Active transport Many models & mechanisms ATP ATP

31. How about large molecules? Moving large molecules into & out of cell through vesicles & vacuoles endocytosis phagocytosis = “cellular eating” pinocytosis = “cellular drinking” exocytosis exocytosis

32. Endocytosis phagocytosis pinocytosis receptor-mediated endocytosis fuse with lysosome for digestion non-specific process triggered by molecular signal

33. 2007-2008 The Special Case of Water Movement of water across the cell membrane

34. Osmosis is diffusion of water Water is very important to life, Diffusion of water from high concentration of water to low concentration of water across a semi-permeable membrane

35. Concentration of water Direction of osmosis is determined by comparing total solute concentrations Hypertonic - more solute, less water Hypotonic - less solute, more water Isotonic - equal solute, equal water hypotonic hypertonic water net movement of water

36. freshwater balanced saltwater Managing water balance Cell survival depends on balancing water uptake & loss

37. RECEPTORS

38. Cell Surface Receptors Can be divided into several categories g b a Ligand-gated Ion channels G-protein coupled receptors Receptor tyrosine kinases a b Integrins Toll-like receptors N C

39. Ligand gated Ion channels Pentamers – 5 subunits Top view subunit pore subunit pore Cell membrane

40. Ligand gated Ion channels Ligands are molecules that act like keys that fit certain binding pockets or locks on the receptor. Activated or turned on by ligands Cell membrane

41. GABA Glycine Nicotinic acetylcholine Serotonin Glutamate Examples of these:

42. Seven pass/ Serpentine receptors

43. G protein Effector pathway Substrates Gs Adenylyl cyclase Beta-receptors, H2, D1,serotonin Gi Adenylyl cyclase Muscarinic M2 D2, alpha-2,opioid Gq Phospholipase C Alph-1, AT1, M1, M3 Go Ca++ channel K+ channel in heart, SM G-protein coupled receptors

44. g b a G-protein coupled receptors Cell membrane G-protein composed of one alpha, beta, and gamma subunit 2 primary signaling cascades: cAMP or phosphatidylinositol pathways Pathway activated depends on alpha subunit type (G α s, G α i /o, G α q/11, G α12/13 ) GDP bound to a when inactive g b a GDP

45. g b a G-protein coupled receptors Cell membrane When a ligand binds, the receptor changes conformation, allowing G-protein to be activated (GDP is exchanged for GTP) G-protein dissociates from receptor then subunits from each other. GDP GTP a GTP

46. g b a cAMP pathway Cell membrane GDP GTP a GTP G α s binds to Adenylate Cyclase (AC) and stimulates cAMP synthesis from ATP G α i/o binds to AC and inhibits cAMP synthesis AC AC ATP cAMP

47. g b a Phosphatidylinositol pathway GDP GTP a GTP G α q/11 binds to Phospholipase C (PLC) and catalyzes the cleavage of phosphatidylinositol 4,5-biphosphate (PIP2) into the second messengers inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). PLC PLC DAG IP3 PIP2 P P P To sarcoplasmic reticulum…

48. EXAMPLES Muscarinic c holinergic receptors M1, M3, M5- Gq M2, M4- Gi Adrenergic receptors α1 - Gq α2 - Gi β 1 - Gs β 2 - Gs β 3 - Gs Dopamine receptors D1 - Gs D2 - Gi

49. GABA receptors GABA- B – Gi TSH receptors - Gs LH receptors ACTH receptors Rhodopsin receptors Oxytocin Neuropeptides Vasopressin V1- Vascular receptor ( Vasocinstriction ) V2- Collecting duct V3- Anterior pituitary AT II VIP

50. Receptor tyrosine kinase Tyr Tyr Tyr Tyr Tyr Tyr Two inactive monomers contain tyrosine (Tyr) residues Ligand binding to the monomers leads to dimer formation Tyr Tyr Tyr Tyr Tyr Tyr Cell membrane

51. Receptor tyrosine kinase Tyr Tyr Tyr Tyr Tyr Tyr ATP ATP ATP ATP ATP ATP P P P P P P ATP molecules donate a phosphate (P) to each of the tyrosines . Inactive relay proteins bind to the phosphorylated tyrosine residues and trigger cellular responses downstream Cellular responses Cell membrane

52. Insulin Vascular Endothelial Growth Factor (VEGF) Platelet-derived Growth Factor (PDGF) Epidermal Growth Factor (EGF) Fibroblast Growth Factor (FGF) Nerve Growth Factor (NGF) Examples of these:

53. JAK STAT Signaling Ligand Binding JAK Activation Cell membrane JAK JAK P P

54. JAK STAT Signaling Phosphorylation of Tyrosine Residues Tyr Tyr JAK JAK P P Cell membrane STAT

55. JAK STAT Signaling Phosphorylation of STAT Tyr Tyr JAK JAK P P Cell membrane STAT STAT P P

56. STAT Cytokine Responsive Gene Gene Transcription Nucleus STAT P P JAK STAT Signaling STAT Translocation to Nucleus

57. Growth Hormone Prolactin IL-2 Cytokines T cell receptors B cell receptors Examples of these:

58. Receptor tyrosine phosphatase

59. Serine Threonine kinase pathway/ B Raf

60. Examples: TGF-Beta BMP B- Raf Inhibitors Sorafenib - Approved for Liver and Kidney cancers vemurafenib  and dabrafenib  are approved by FDA for treatment of late-stage melanoma.  Trametinib - FDA-approved to treat BRAF-mutated melanoma.

61. ANP receptors

62. Integrins Cytoskeleton a b Extracellular matrix (ECM) Join the cytoskeleton on the inside of the cell to the extracellular matrix on the outside. Heterodimers of alpha and beta subunits Cell membrane

63. Toll-like receptors nucleus Cell membrane DNA Involved in the immune response Signals between downstream proteins result in enhanced transcription of inflammatory genes Immune response

64. Disorders Some disease resulting from or attributed to abnormalities of Membranes

65. Disorder due to defect in ion channels

66. Some drugs acting on cell membrane

67. Local anaesthetics

68. Polymyxin B & Colistin They are active against gram-negative bacteria only. Colistin is more potent on Pseudomonas, Salmonella and Shigella . Rapidly acting bactericidal agents They have high affinity for phospholipids: the peptide molecules (or their aggregates) orient between the phospholipid and protein films in gram-negative bacterial cell membrane causing membrane distortion or pseudopore formation →ions, amino acids, etc. leak out.

69. Given orally, side effects are limited to the g.i.t . Systemic toxicity of these drugs (when injected) is high: flushing and paresthesias (due to liberation of histamine from mast cells), marked kidney damage, neurological disturbances , neuromuscular blockade . Uses: skin infections, burns, otitis externa , conjunctivitis, corneal ulcer—caused by gram-negative bacteria including Pseudomonas. Gram-negative bacillary (E. coli, Salmonella, Shigella ) diarrhoeas, especially in infants and children

70. Daptomycin Lipopeptide antibiotic used in the treatment of systemic and life-threatening infections caused by Gram-positive organisms Inserts into the cell membrane →aggregates → alters the curvature of the membrane → creates holes that leak ions → rapid depolarization → loss of membrane potential → inhibition of protein, DNA, and RNA synthesis → bacterial cell death . Use: Skin and skin structure infections, MRSA Dose: 4 mg/kg IV

71. Cell membrane active Antifungals