7. membran 2010

Biomolecular Biomolecular Membrane Membrane

Dra Sri Musta’ina, MKes

Functions of Membrane

 1. Compartmentalization

 2. Selectively permeable membrane barrier

 3. Transport phenomena

4. communication and compartment attachment

5. Cell identity and antigenicity

6. Conductivity

The Fluid Mosaic Model

Sheet-like non-covalent assemblies Regulates transport of small and macro-

molecules Controls flow of information between cells

Specific receptors for external stimuli Generation of signals

Cell adhesion for tissue formation. Energy conversion processes- a battery.

- Small platform, composed of sphingolipids, chol & prot

- fluid, more ordered & tightly packed.

- function : regulate membrane functions- Prot : GPI anchored, tyrosin kinase, G

protein fragments, eNOS- Disease : alzheimer, muscular disthrophi,

astme, allergic respons, etc.

Raft clustering.

Transport l I ntas membran

Lipid bilayer

Small hydrophobic molecules: O2, CO2, N2, benzeneSmall uncharged polar

molecules: H2O, ethanol, glycerolLarger uncharged polar

molecules: glucose, amino acid, nucleotides

Plasma membrane is a semi-permeable membrane

Ions: H+, Na+, HCO3- ,

K+, Ca+, Mg2+, Cl- , etc.

Overview of transport mechanismsOverview of transport mechanisms

DIFFUSION (hydrophobic/ lipophilic molecules only): a slow process.

FACILITATED DIFFUSION, uniport (e.g. glucose transporters)

CO-TRANSPORT: symport (same direction) or antiport (opposite directions

ACTIVE TRANSPORTER, against the concentration gradient

CHANNELS, e.g. sodium channel, water channel (aquaporin)

ATP

ADP + Pi

Diffusion : kinetic energy

Diffusion rate: temperature distance

concentration gradienareaDetermined by : molecular size + attribute

solvent characteristic

Simple diffusion : - oxygen

- carbon dioxide- water- uncharged molecules- lipid soluble molecules

OSMOSIS• diffusion of water molecules through a

selectively semipermeable membrane• from hypoosmotic sol to hyperosmotic sol• continues until : equilibrium reached cell destroyed stopped by opposing force

SOLVENT FLOW. Movement of water occurs by one of two

processes.

1. Osmosis

2. Filtration – (Example: urine formation in kidney)

3. Specific Channel

Passage of water molecules through the aquaporin AQP1. Because of the positive charge at the center of the channel, positively charged ions such as H3O+, are deflected. This prevents proton leakage through the channel.

AquaporinAquaporin water channel proteins called Aquaporins (AQP1 & AQP2)

Peter Agre’s experiment with cells containing or lacking aquaporin. Aquaporin is necessary for making the 'cell' absorb water and swell.

Facilitated diffusion

Molecules with low permeability coefficients can go through the membrane faster than normal diffusion process.

Ex : uptake of glucose into erythrocyte. It is rapidly moved across the membrane down

the concentration gradient with the help of a membrane protein called permease.

The velocity of transport is saturablePermease is a multipass transmembrane

protein to facilitate the diffusion of specific molecules across biological membrane.

Facilitated Diffusion: with molecule specific permease

Glucose transporter (permease)

ATP ADP

Transport velocity

External concentration of glucose, mM

Simple diffusion

Facilitated diffusion

Vmax

Half Vmax

Km

Rate

of G

luco

se U

ptak

eTransporters must have a specific binding site for the solute, e.g. glucose. Once they get in the cells, they are usually phosphorylated to go into metabolic pathway and hence the outer concentration of glucose is higher than inside.

Saturable

Facilitated Facilitated DiffusionDiffusion of Ions of IonsThe transmembrane channels that permit facilitated diffusion can be opened or closed. They are said to be "gated”

1. Ligand-gated ion channels. open or close in response to binding a small signaling molecule or

"ligand". (extracellular ligands and intracellular ligands) The ligand is not the substance that is transported when the channel opens.

2. Mechanically-gated ion channels Examples: Mechanical deformation of the cells of stretch receptors

opens ion channels leading to the creation of nerve impulses.

3. Voltage-gated ion channels In so-called "excitable" cells like neurons and muscle cells, some

channels open or close in response to changes in the charge (measured in volts) across the plasma membrane.

ChannelsChannelsVoltage gated

channels

Sodium channel consists of FOUR transmembrane domain, each has SIX transmembrane αhelices, the forth helice is believed to be the voltage sensor.

Potassium channel has ONE molecule of only SIX transmembrane α helices

COOH

COOH

NH

2

NH2

Active transport

Use ATP hydrolysis directly or indirectly (secondary active transport) to move molecules across the membrane against the concentration gradient.

Active transporters are membrane proteins specifically bind and move the molecules across the membrane to a unique direction using ATP hydrolysis as an energy source.

Mainly for ions and chemicals, creating a chemical gradient using ATP energy.

The gradients created maintain many life processes..

Concepts of active transport

TRANSPORTER• specific• conformation changes• slower than Channel protein• 3 tipe : - passive transporter - active transporter - group transporter- Mechanism : - uniporter - antiporter - symporter

Tipe TransporterUNIPORTER1 molekul, searah gradien konsentrasic/ : glukosa transporter

SYMPORTER• tanpa ATP, energi dari gradien • melibatkan 2 jenis molekul • sama dengan kerja pompaarah : sama, c/ : Na+ dan glukosa, di lumen usus-jaringanANTIPORTERarah : berlawanan c/: Na+ dan Ca+, di otot jantung

P-class pump Perubahan konformasi protein yang disebabkan karena proses fosforilasi oleh ATP

F-class / V-class Proton transporting protein (H+).V-class : mempertahankan pH, pada lisosom, dan vesikula lainF-class : di mitokondria, melawan gradien elektrokimia

Indirect active transport

Three types of ATPases: P, V & F.

α αβ β

P-type ATPase

V-type ATPase F-type ATPase

F1 complex

F0complex

V1 complex

V0 complex

1997 Nobel prize of Physiology and Medicine went to Paul Boyer and John Walker for their work on ATP synthase (F type), and Jens Skou for his work on Na-K-ATPase, which uses one-third of the ATP made by ATP synthase.

ATP synthaseProton

pump

P-type: Na+-K+-ATPase, Ca2+ and H+ pump, P means they have phosphorylation and they all sensitive to inhibition.

V-type: inner membrane ATPase to regulate H+ and adjust proton gradients, v means vacuole type for acidification of lysosomes, endosomes, golgi, and secretory vesicles.

F-type: ATP synthase to generate ATP energy from moving the proton across; F means energy coupling factor. There are F1 and F0 subcomplexes: F1 generates ATP, F0 lets H+ go through the membrane.

ABC transporters: ATP-binding cassette protein for active transport of hydrophobic chemicals and Cl-.

Classified according to their protein sequence homology and structures.

P-type ATPase: Ca2+ATPase

Other divalent ion transporters have similar structure with this Ca2+-ATPase and the αsubunit of Na+-K+-ATPase.

NH2

COOH

Ca2+ binding siteAspartate

ATP binding site

ATP

ADP

Ca2+

ABC (ATP-Binding Cassette) transporters: 6 or 12 trans-membrane helices with 2 ATP binding sites, drug or ligand- binding sites yet to be clearly identified.

ATP binding domains

NH

2 COOH

Oligosaccharide chains

P-glycoprotein

R domain

NH

2

ATP binding domains

COOH

Chloride channel: the cystic fibrosis transmembrane conductance regulator,

CFTR, has an extra R domain

Vesicle-mediated transport

Vesicle-mediated transport

- receptor-mediated endocytosis: requires specific receptor/ligand pair

- small pit contains ligand receptors exposed to extracellular space and adaptin and clathrin on the intracellular side (skeleton for vesicle)

- adaptin links receptor (intracellular side) and clathrin to form a coated pit

- clathrin forms polyhedral cage giving the "coat" of the vesicle

- pinching off of vesicle into cell is requires dynamin,a GTPase

- clathrin-coated vesicle is "de-coated" of clathrin and the proteins involved in formation are recycled while the vesicle is further processed

- phagocytosis:ingestion of particles (bacteria, dust, carbon particles)

Vesicular Transport : Receptor Mediated Endocytosis

Junction

A. CELL TO CELL JUNCTION. 1. OCCLUDING JUNCTION / TIGHT JUNCTION 2. ADHERING JUNCTION : actin filament = ZONULLA intermediate filament =

MACULAB. CELL TO CELL COMMUNICATION : gap junctionC. CELL – MATRIX / ANCHORING JUNCTION Not only hold cells together but provide tissues with structural cohesion. These

junctions are most abundant in tissues that are subject to constant mechanical stress such as skin and heart.

1. FOCAL ADHESION / DESMOSOME plasma membrane - plasma membrane of adjacent cells.

2. HEMI DESMOSOME cytoskeleton and extracellular matrix components such as the basal laminae that underlie epithelia

Occluding junction : tight junction

Adhering junction :

a. Zonula adherent

b. Macula adherent

Gap junction

Cell – cell communication

Cell-matrix Adhessive junction : anchoring junction

Desmosome hemidesmosome

Anion antiport in parietal cells of stomach with H+- K+ -ATPase to produce stomach acid.

Basolateral membrane

Apical membrane

CO2CO2

HCO3

Carbonic anhydrase

Cl - Cl -Cl -

HCO3

K+

K+

K+ channel

Anion antiportCl -channel

H+-K+-ATPaseATP

ADP + Pi

K+H2O

+ OH

-

Omeprazole inhibits the proton pump.

Omeprazole and Cimetidine stop stomach acid

• H+-K+-ATPase is an electroneutral antiport. K+ is removed by K+ channel and concurrently Cl- channel removes Cl- to the same direction.

• HCl is the overall transport product in the stomach lumen.

• Omeprazole inhibits the proton pump directly.• Cimetidine resembles histamine to block the

binding of histamine to its receptor thus inhibit the activation of H+K+-ATPase by histamine receptor.

Glucose uptake from lumen to the capillaries using glucose transporter (permease), glucose-sodium symport and Na+-K+-ATPase.

Glucose gets into the cell and

simultaneously transported by

glucose transporter into the capillaries.

Na+Na+Na+

K+K+Na+-Glucose Symport

ATP

ADP + Pi

Na+-K+-ATPase

Glucose transporter: permease

Intestinal lumen

Capillaries

Glucose-NaGlucose-Na++ symport is driven symport is driven

by intracellular by intracellular Na+ levels using Na+ levels using

Na+ –K+ -ATPase. Na+ –K+ -ATPase.

Primary and secondary active transporters work coordinately in animal cells. They generate membrane potential, generate proton gradient, maintain acidity, etc…..

K+

K+

Na+

Na+

Na+

Na+ -driven symport

soluteNa+

soluteNa+ -K+ ATPase

ATP

ADP + Pi

ATP

H+

H+

H+ ATPase

K+ = 140 mMNa+ = 12 mM

K+ = 4 mMNa+ = 145 mM

----

++++

ADP + Pi

Co-transport Systems and Co-transport Systems and their couplingtheir coupling

Adapted from Sperlakis (ed)., 1998. Cell Physiology Source Book. Academic Press.

Na+Na+

Ca-Na Antiport

Ca-ATPase

Ca-channel

Na-K ATPase

Sarcoplasmic reticulum

K+

Na+-K+-ATPase and Ca2+-ATPaseNa+-K+-ATPase and Ca2+-ATPase

Ouabain inhibits Na-K-ATPase.

Ouabains for treatments of angina pectoris and myocardial infarction

Ouabain blocks Na + -K + -ATPase. By blocking the Na+-K+-ATPase, Intracellular Na+ remains high

Hence, the Na+- Ca2+ antiport cannot remove Ca2+ ions out from the cardiac muscle cells.

Eventually, the Ca2+ ion level is restored to maintain the contraction power of cardiac muscle.

MEMBRANE & DISEASES

Can be disrupted by :- Change to protein- component of the lipid bilayer : protein lipid composition- component of cytoskeleton-

Categories of “true” membrane based disease :

1.Defects in cytoskeletal component impair membrane function

2.Altered membrane lipid composition disrupts membrane traficking.

Defects in cytoskeletal component and membrane function :

Ex.-Sickle cell anemiaActin/spectrin lattice “lock”, RBC less drformable and obstruct the microcirculation

- Duchene muscular dystrophyMutation in dystrophin gene disrupts the ability of protein product to anchor cytoskeletal element to the surface membrane. Structural support is loss, membrane becomes permeable, intracell pressure ￪ and cell explodes.

DisrupTs of membrane trafficking

-Rab protein : Lipid modification to carboxyl terminus.

-Hermansky-Pudlak, Griscelly sindrome Lysosome-related organelle

-Niemann-Pick Disease type C (NPC) LSD

Protein membran dysfunctionProtein membran dysfunctionCystic fibrosisDefect pada mekanisme transport chlorida di epitel

paru(CFTR = Cystic Fibrosis Transmembrane Regulator)

Pathologic condition : CFTR transport Cl- : epithelial – airway lumen,

followed by Na+ and water : thick & dehydrated mucous in the lungs

Good breeding ground for infection by bacteria Trouble breathing

Intracellular drug delivery

- Uptake by cell : 1. endocytosis

~ 500 nm

clathrin coated pits

pH environment

2. macropinocytosis, phagocytosis

scavenger (mØ, neutrophils) APC

size : up to the size of cell

- Cross the plasma membrane

- Escape from endosome/lysosome

- Cross the plasma membrane Direct entry to cytosol Viral peptide transporters

- Escape from endosome/lysosome cargo released from vesicle once taken inside the cell routes : - viral peptides evolved for endosomal escape - fusion with endosomal membranes - disruption of endosomal compartments

Intracellular drug delivery (cont)