CELL MEMBRANE PHYSIOLOGY &PHARMACOLOGY

Dr Shahid SaacheDept. of Pharmacology

BJ GMC, PuneMentor- Dr Sujeet

Divhare

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

Fungal cell membrane

Why cell membrane is important to study??

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.

COMPOSITION OF CELL MEMBRANE

1. Lipids• Phosopholipids• Sterols

2. Proteins• Integral• Peripheral

3. Carbohydrates• Glycolipids• Glycoproteins

Phospholipids

Fatty acid

Phosphate

Fatty acid tails hydrophobic

Phosphate group head hydrophilic

Arranged as a bilayer

Phospholipid bilayer

polarhydrophilic

heads

nonpolarhydrophobic

tails

polarhydrophilic

heads

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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Fluidity of membrane

• Important for exocytosis and endocytosis• For membrane biogenesis

Factors altering fluidity• Temperature ↑….. Fluidity• Cholesterol content ↑….. Fluidity

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Role of Fluidity of membrane

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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!

Membrane is a collage of proteins & other molecules embedded in the fluid matrix of the lipid bilayer

Extracellular fluid

Cholesterol

Cytoplasm

Glycolipid

Transmembraneproteins

Filaments ofcytoskeleton

Peripheralprotein

Glycoprotein

Phospholipids

Membrane proteins

• Classified depending on type of interaction with bilayer.

i. Integral membrane proteins- pass through bilayer

ii. Peripheral membrane proteins- associate with bilayer by non-covalent interactions

iii. Lipid-anchored proteins.

Lipid-anchored membrane proteins

• Covalently linked to membrane by short oligosachharide linked to a molecule of GPI embedded on outer leaflet.

• Example –Scrapie protein PrPc

• Some linked to inner leaflet like Ras and Src proteins have been implicated in transformation of normal cell to malignant cell.

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

Six major functions of membrane proteins

Transport Enzymatic activity Signal transduction

Cell-cell recognition Intercellular joining Attachment to thecytoskeleton ECM

•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

Movement across the Cell Membrane

Diffusion 2nd Law of Thermodynamics

governs biological systems universe tends towards disorder (entropy)

Diffusion movement from high low concentration

Diffusion Move from HIGH to LOW concentration

“passive transport” no energy needed

diffusion osmosis

movement of water

Diffusion across cell membrane Cell membrane is the boundary between

inside & outside… separates cell from its environment

INfoodcarbohydratessugars, proteinsamino acidslipidssalts, O2, H2O

OUTwasteammoniasaltsCO2

H2O products

IN

OUT

Diffusion through phospholipid bilayer What molecules can get through directly?

fats & other lipids

inside cell

outside cell

lipidsalt

aa H2Osugar

NH3

What molecules can NOT get through directly?

polar molecules H2O

ions salts, ammonia

large molecules starches, proteins

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

Channels through cell membrane Membrane becomes semi-permeable

with protein channels specific channels allow specific material

across cell membrane

inside cell

outside cell

sugaraaH2O

saltNH3

Facilitated Diffusion Diffusion through protein channels

channels move specific molecules across cell membrane

no energy neededopen channel = fast transport

facilitated = with help

high

low

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

symportantiport

Active transport Many models & mechanisms

ATP ATP

How about large molecules? Moving large molecules into & out of cell

through vesicles & vacuoles endocytosis

phagocytosis = “cellular eating” pinocytosis = “cellular drinking”

exocytosis

exocytosis

Endocytosis

phagocytosis

pinocytosis

receptor-mediated endocytosis

fuse with lysosome for digestion

non-specificprocess

triggered bymolecular signal

2007-2008

The Special Case of Water

Movement of water across the cell membrane

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

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

freshwater balanced saltwater

Managing water balance Cell survival depends on balancing

water uptake & loss

RECEPTORS

Cell Surface ReceptorsCan be divided into several

categories

gb

aLigand-gated

Ion channel

sG-protein coupled

receptors

Receptor tyrosine kinases

a b

IntegrinsToll-like

receptors

N

C

Ligand gated Ion channels

Pentamers – 5 subunits

Top view

subunitpore

subunit

pore

Cell membrane

Ligand gated Ion channelsLigands are molecules that act like keys that fit certain binding pockets or locks on the receptor.

Activated or turned on by ligands

Cell membrane

GABAGlycineNicotinic

acetylcholineSerotoninGlutamate

Examples of these:

Seven pass/ Serpentine receptors

G protein Effector pathway Substrates

Gs Adenylyl cyclase Beta-receptors, H2, D1,serotonin

Gi Adenylyl cyclase Muscarinic M2D2, alpha-2,opioid

Gq Phospholipase C Alph-1, AT1, M1, M3

Go Ca++ channel K+ channel in heart, SM

G-protein coupled receptors

g

ba

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• (Gαs, Gαi/o, Gαq/11,

Gα12/13)• GDP bound to a when

inactive

GDP

g

ba

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

aGTP

g

ba

cAMP pathway

Cell membrane

GDP

GTP

aGTP

• Gαs binds to Adenylate Cyclase (AC) and stimulates cAMP synthesis from ATP

• Gαi/o binds to AC and inhibits cAMP synthesis

AC

ATP

cAMP

g

ba

Phosphatidylinositol pathway

GDP

GTP

aGTP

• Gαq/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).

PLCPLC

DAG

IP3

PIP2

P P

P

To sarcoplasmic reticulum…

EXAMPLES

• Muscarinic cholinergic receptors• M1, M3, M5- Gq• M2, M4- Gi

•Adrenergic receptors• α1 - Gq• α2 - Gi• β1 - Gs• β2 - Gs• β3 - Gs

•Dopamine receptors• D1 - Gs• D2 - Gi

• GABA receptors– GABA- B – Gi

• TSH receptors- Gs• LH receptors• ACTH receptors• Rhodopsin receptors• Oxytocin

• NeuropeptidesVasopressin – V1- Vascular receptor

(Vasocinstriction)– V2- Collecting duct– V3- Anterior pituitary

• AT II• VIP

Receptor tyrosine kinase

TyrTyrTyr

TyrTyrTyr

• Two inactive monomers contain tyrosine (Tyr) residues

• Ligand binding to the monomers leads to dimer formation

TyrTyrTyr

Cell membrane

Receptor tyrosine kinase

TyrTyrTyr

TyrTyrTyr

ATPATPATP

ATPATPATP

PPP

PPP

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

InsulinVascular Endothelial

Growth Factor (VEGF)Platelet-derived

Growth Factor (PDGF)Epidermal Growth

Factor (EGF)Fibroblast Growth

Factor (FGF)Nerve Growth Factor

(NGF)

Examples of these:

JAK STAT Signaling

Ligand BindingJAK Activation

Cell membrane

JAKJAK PP

JAK STAT Signaling

Phosphorylation of Tyrosine Residues

Tyr Tyr

JAKJAK PP

Cell membrane

STAT

JAK STAT Signaling

Phosphorylation of STAT

Tyr Tyr

JAKJAK PP

Cell membrane

STATSTAT P P

STAT

Cytokine Responsive Gene

Gene Transcription

Nucleus

STATP P

JAK STAT Signaling

STAT Translocation to Nucleus

Growth HormoneProlactinIL-2CytokinesT cell receptorsB cell receptors

Examples of these:

Receptor tyrosine phosphatase

Serine Threonine kinase pathway/ B Raf

Examples:•TGF-Beta•BMPB-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.

ANP receptors

Integrins

Cytoskeleton

a bExtracellular 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

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

DisordersSome disease resulting from or attributed to abnormalities of Membranes

Disorder due to defect in ion channels

Some drugs acting on cell membrane

Local anaesthetics

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.

• 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

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

Cell membrane active Antifungals