Plasma+Membrane+II


 * 23 August 2006**
 * Plasma Membrane II**
 * Dr. Cynthia Smas, Ph.D.**

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=**Overview**=


 * Composition of extracellular and intracellular fluids differs greatly due to selective permeability of the plasma membrane
 * Solute concentration differential drives diffusion of solute
 * Can be utilized as a source of energy to drive secondary active transport
 * Without selective permeability, extracellular fluid (ECF) would be the same as intracellular fluid (ICF)

**Lipid Bilayers**

 * Selectively maintains differences in ECF and ICF
 * Some molecules and pass directly through the lipid core
 * Others must use membrane proteins to serve as structures for passage
 * Channels, transporters, junctions, pumps

**Cell signaling**

 * Communication of external signals occurs either directly across cytoplasm to cytoplasm or indirectly
 * Extracellular molecules pass through the plasma membrane via specific membrane proteins

=**Osmosis**=


 * Water flows across the plasma membrane in the direction that would lead to the equalization of solute concentrations across the membrane
 * Water can also flow through aquaporins – specialized channels
 * Isotonic – equal osmotic pressure
 * Hypotonic – osmotic pressure flows into cell
 * Hypertonic – osmotic pressure flows out of cell

=**Facilitated Diffusion**=


 * Rate of facilitated diffusion is much higher than passive diffusion.
 * Transport is specific and occurs via limited number of proteins.
 * Accelerate reaction that is already thermodynamically favored (similar to enzymes)
 * No ATP necessary.
 * Utilizes binding to specific membrane proteins (transporters) which are carrier-mediated and behave kinetically.
 * Facilitated diffusion can be saturated because of limited number of transporters
 * Example: GLUT proteins and cellular glucose uptake
 * 12 transmembrane α-helices
 * Transport direction dependent on transporter conformation and relative substrate concentrations.

**GLUT4 Protein**

 * Insulin responsive glucose transporter – insulin action causes rapid increase in glucose uptake
 * Extracellular insulin binds to plasma membrane receptor
 * GLUT4 is translocated from a pre-formed intracellular pool
 * GLUT4-containing vesicles fuse with the plasma membrane, increasing GLUT4 on the cell surface
 * Defects can lead to insulin resistance and type II diabetes.

**Ion Channel-Mediated**

 * Cells must maintain electrochemical gradients to function
 * Gradients are a source of energy for driving many cellular processes
 * Ion channels are a form of facilitated diffusion
 * Ions can also enter by active transport via ion pumps
 * Transient alteration of ion membrane permeability can control cell signaling
 * Ion channel can pass 107 to 108 ions/second

**Ion Concentration and Charge Gradients**

 * Ion concentration varies on exterior and interior of cell
 * Concentrations contribute to the total charge differential across the membrane
 * Sum of free energy change determines direction and rate of ion flow
 * ΔG of ion concentration gradient plus the ΔG from electrical gradient equals the total ΔG
 * Most Ion channels are gated and operate in response to specific signals
 * i.e. voltage-gated or ligand-gated channels
 * Exception: K+ leak channels are always open and act to maintain the negative membrane resting potential (cytoplasm negative).
 * White ion channel opening responds to specific signals, the direction and flow rate relies on the relevant electrochemical gradient across the plasma membrane.

**Ligand-Gated Ion Channels**

 * Example: Nicotinic Acetylcholine Receptor – allows passage of Na+ and K+.
 * Nicotinic acetylcholine are at the neuromuscular junction (NMJ)
 * Large flux of Na+ into the cell and K+ can enter, resulting in a transient depolarization of the membrane
 * Leads to an action potential necessary for muscle contraction.
 * Uses a rotation and sliding of helices to open and close the ion channel

**Voltage-Gated Ion Channels**

 * Important for nerve impulse action potentials
 * Multiple related sets of α-helices will come together to form a selective ion pore.
 * Uses the ball-and-chain mechanism to inactivate the channel
 * Channel is closed when polarized
 * When depolarized, the channel opens
 * After depolarization occurs, the “ball” part of the protein inactivates the channel to stop depolarization
 * After repolarization, the channel closes and the “ball” returns to original conformation.

**Selectivity of Ion Channels**

 * Selectivity of ion channel depends on the structure of the channel (the amino acids in the selectivity filter)
 * All ions exist in a hydrated form with a distinctive water shell
 * Ion enters up to the point of the ion channel known as the selectivity filter.
 * At the selectivity filter, the channel pore size becomes too small for a hydrated ion to pass
 * The ion must shed its water shell in a thermodynamically favored manner, i.e. other favorable polar interactions must replace those the ion had with water.
 * For a K+ channel, K+ is thermodynamically favored to pass but Na+ is not.

**Rate of Transport**

 * Two-site model has two binding sites in a channel’s selectivity filter with the first ion reaching the second binding site
 * The next ion that binds to the first binding site and creates an electrostatic repulsion that pushes the first ion out
 * This process repeats to allow efficient transport of ions.

=**Active Transport**=


 * Require energy input to function against the electrochemical gradient.
 * If the transporter protein itself hydrolyses ATP, it is called primary transport
 * If unfavorable (uphill) flow of one molecule is coupled to a favorable (downhill) flow of another, this is called secondary transport

**Primary Transport**

 * Three families of primary active transporters: P-type, ABC-type, and F&V-type
 * F&V-type pumps only H+ and is common in bacteria and plants

**P-Type Primary Transport**

 * ATP-powered pumps such as Ca2+ ATPase in muscle SR, H+/K+ ATP in the stomach, Na+/K+ ATPase in all cells, among others.
 * Hydrolysis of ATP provides energy to pump against the electrochemical gradient
 * Phosphoryl group of ATP becomes covalently bound to the transporter
 * Requires Mg2+ cofactor
 * Two integral protein subunits
 * Example: Na+/K+ pump generates an ion concentration gradient for controlling:
 * Cell volume
 * Driving transport of sugars and amino acids
 * Establishing and maintaining electrochemical gradients
 * Maintaining membrane charge potentials at -60mV with K+ leak channels.
 * 25%-75% of all cytosolic ATP present in cells is hydrolyzed by action of the Na+/K+
 * Distinct conformational states (E1 and E2) and phosphorylation state governs transport.
 * Cardiotonic steroid drugs (Digitalis) act by inhibiting the function of the Na+/K+ ATPase where the dephosphorylation step occurs
 * This locks the pump into a non-functional state, decreasing pump action and increasing Na+ in cardiac muscle cells
 * This leads to increase Ca2+ in the cell via action of Na+/Ca2+ transporter
 * Ca2+-mediated signals act to increase contraction strength of the heart muscle

**ABC-Type Primary Transport**

 * **A**TP-**B**inding **C**assette Primary Transporter
 * Transports ions and small molecules
 * Has 6 α-helices form transmembrane domain and is structurally and functionally different than P-type pumps.
 * Functional domains (1-4) of ABC-transporters may be single or separate units.
 * ATP hydrolysis is coupled with solute movment
 * Example: MDR-1 and chemoresistant cancers
 * Small planar drugs diffuse into cells
 * Cell sees that drug as toxic and MDR-1 uses ATP to export drug from cytosol
 * Drugs fail to exert benefits and tumor cells become chemoresistant to multiple drugs simultaneously and lead to uncontrollable tumor growth.
 * MDR-1 gene is frequently amplified in multi-drug resistant cancer cells
 * MDR-1 inhibitors in clinical trials seems promising
 * MDR-1 is also found in normal tissues especially in the liver to get rid of toxins
 * Example: CFTR chloride channel and cystic fibrosis
 * Cystic fibrosis is a autosomal recessive genetic disease of mucus glands, affecting respiratory and digestive systems.
 * deltaF508CFTR – Deletion of 3 bp in CFTR
 * Mutant protein does not reach the cell surface and cannot function likely due to protein misfolding and inability for molecular chaperone to bring it to the cell surface.
 * Gene therapy is currently investigated to help the mutated deltaF508CFTR reach the cell surface and provide some relief to cystic fibrosis patients.
 * CFTR is present on apical surface of epithelial cell plasma membrane where it pumps Cl- out of the cell
 * deltaF508CFTR mutated cells accumulate Cl- in the cell, causing Na+ and water to come in from the extracellular space
 * Loss of water from extracellular space results in thick, dehydrated mucus and defective function of the respiratory tract cilia and increased infections.

**Secondary Transport**

 * Use one concentration gradient to power another gradient
 * Example: Na+/Glucose symport
 * ATP is used to pump Na+ to generate a Na+ concentration gradient
 * By coupling the Na+ downhill flow with the uphill of glucose, glucose can overcome its gradient and move into a cell
 * Antiporter – flow of A moves in the opposite direction of B
 * Symporter – flow of A moves in the same direction of B

=**Integration of Membrane Selective Permeability**=


 * Example: Glucose Transport by Intestinal Epithelia
 * Oral rehydration therapy depends on the function of Na+/Glucose Symporter
 * Both NaCl and Glucose is necessary because the symporter needs both to function
 * Transport of glucose and NaCl across the intestinal epithelium causes water from the intestinal lumen to move into the blood, leading to rehydration

=**Signal Transduction**=


 * Molecule does not actually enter a cell, just the signal such as by second messengers
 * Extracellular signal may be soluable (i.e. hormone or steroid) or on the plasma membrane of another cell
 * Endocrine – in the circulation
 * Paracrine – from an adjacent or nearby cell
 * Autocrine – from that cell itself

**Stages of Signal Transduction**

 * Signal – extracellular signal
 * Reception – received on the plasma membrane
 * Transduction – Occurs in the cell interior where receptor is phosphorylated and signaling cascades may occur
 * Enzymes central to signal transduction are located at the plasma membrane
 * Example: Adenylate Cyclase – generates cAMP from ATP
 * Example: Phospholipase C – generates DAG and IP3
 * Various signal transduction events are integrated for proper function, i.e. signal transduction has a lot of “cross-talk.”
 * Response – response to the signal by gene regulation, phosphorylation, etc. and can cause many levels of positive or negative feedback

**Secondary Messengers**

 * Initial signal (first messenger) is amplified and propagated by a second messenger in side the cell
 * Examples: cAMP, cGMP, Ca2+, Inositol 1,4,5-triphosphate (IP3), Diacylglycerol (DAG)

=**Objectives**=