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TRANSPORT ACROSS MEMBRANES Associate Professor Dr. Wipa Suginta School of Biochemistry, Institute of Science Membrane transport_WipaSuginta 1/2556 1 Types of transport across membranes Transporting process Transporter Passive - Simple diffusion - Facilitated diffusion None Gap junctions Ion channels Carriers: glucose transporter Active -Primary active transport -Secondary active transporter Pumps: Na+/K+ pump Antiporters/Symporters Membrane transport_WipaSuginta 1/2556 2 Types of membrane transporter Membrane transport_WipaSuginta 1/2556 3 General Diffusion 1. Downhill process. 2. No energy required. 3. Extremely slow. Membrane transport_WipaSuginta 1/2556 4 Diffusion depends on permeability of molecules (P) across a membrane which can be expressed as P= KD d P = permeability coefficient (cm/sec) K = partition coefficient D = diffusion coefficient (cm2/sec) d = width of the cell membrane (cm) Membrane transport_WipaSuginta 1/2556 5 Effects of molecular weight and solubility on diffusion coefficient Diffusion in water Diffusion in lipid membranes Membrane transport_WipaSuginta 1/2556 6 Membrane permeability in weak acids and bases Membrane transport_WipaSuginta 1/2556 7 When molecules diffuse, free energy is stored in concentration gradients The free energy can be given as: ΔG = RT ln (c2/c1 ) = 2.303 RT log10(c2/c1 ) R = gas constant (8.315 x 10-3 kJ mol-1 or 1.987 x 10-3 kcal mol-1 ) T = temperature (kelvin, K) c = the concentration of the transported solutes Membrane transport_WipaSuginta 1/2556 8 Transport of charge species across membrane generating the electrical membrane potential ΔG = RT ln (c2/c1 ) + ZFV = 2.303 RT log10(c2/c1 ) + ZFV R = gas constant (8.315 x 10-3 kJ mol-1 or 1.987 x 10-3 kcal mol-1 ) T = temperature (kelvin, K) c = the concentration of the transported solutes Z = the electrical charge of the transported species F = the Faraday constant (96.5 kJ V -1 mol-1or 23.1 kcal V -1 mol-1 ) V = the potential in volts across the membrane Membrane transport_WipaSuginta 1/2556 9 The magnitude of electrical potential Eion (volts) is given by the Nernst equation ENa (∆V) = RTln(Nal)/Nar) ZF ENa = 0.059log10(Nal)/(Nar) R (the gas constant) = 1.987 cal/(degree · mol), or 8.28 joules/(degree · mol); T (the absolute temperature) = 293 K at 20 °C, Z (the valency) = +1, F (the Faraday constant) = 23,062 cal/(mol · V) or 96,000 coulombs/(mol · V). The ∆G of ion transport is given by ∆G = RTlnc2/c1 + ZFV Z is the electrical charge of the transported species ΔV is the potential in volts across the membrane = 2.303 RT log10c2/c1 + ZF∆V F is the faraday [23.1 kcal V-1 mol-1 (96.5 kJ V-1 mol-1)] Membrane transport_WipaSuginta 1/2556 10 Energy changes accompanying passage of the hydrophilic solute through the lipid bilayer Membrane transport_WipaSuginta 1/2556 11 Facilitated diffusion across membranes can be achieved by protein carriers • Down-concentration gradient • No energy required • Non-specific or specific • Not as slow as general diffusion • Protein mediated •. Inhibitable Membrane transport_WipaSuginta 1/2556 12 Example 1: passive diffusion through gap junction Small molecules of < 1200 Da pass through gap junctions Membrane transport_WipaSuginta 1/2556 13 Kinetics of general diffusion through membranes via non-specific mediators DKA J= × ΔC d D = diffusion constant of solute K = partition coefficient A = area of membrae D = thickness of membrane Membrane transport_WipaSuginta 1/2556 14 Example 2: Diffusion of antimicrobial agent through a general diffusion porin Membrane transport_WipaSuginta 1/2556 15 Diffusion through membranes using a specific mediator Example 1: Glucose Transporters (GLUTs) or glucose permeases Membrane transport_WipaSuginta 1/2556 16 Notkins, JBC, 2002 Model of the mechanism by GLUT1 is a shuttle between two conformational states The glucose transporter has 12 transmembrane a helices. Membrane transport_WipaSuginta 1/2556 17 A ping-pong model of GluT1 that exists in two conformational states 1) State "pong”; the binding sites for solute A are exposed on the outside of the bilayer. 2) State "ping”; the same sites are exposed on the other side of the bilayer. Membrane transport_WipaSuginta 1/2556 18 Kinetics of specific carriers -The process resembles an enzymesubstrate reaction. - Each type of carrier protein has one or more specific binding sites for its solute. - When the carrier is saturated the rate of transport is maximal (Vmax). - Each carrier protein has a characteristic binding constant for its solute, KM = [s]; v = vmax/2. Glc out + GlUT Vmax Km [Glc-GLUT] Glc in + GLUT Rate (v) = Vmax 1+ Km [C] C is the concentration of Sout (initially, the concentration of Sin = 0); Vmax is the rate of transport if all molecules of the transporter contain a bound S, which occurs at high Solute concentrations; and Membrane transport_WipaSuginta 1/2556 Km is the substrate concentration at which half-maximal transport occurs across the membrane. 19 Diffusion through ion channels Ion channels mediate the passage of ions across plasma membranes. • Down-concentration gradient • No energy required • Rapid diffusion • Selective • Protein mediated (ion channel) • Gating controlled by ligand/voltage • Inhibitable Membrane transport_WipaSuginta 1/2556 20 Membrane transport_WipaSuginta 1/2556 21 The rate of ion transport is influenced by the ion concentrations and by the voltage (i.e., the electric potential). Experimental system for generating a transmembrane voltage potential across a membrane separating a 150 mM KCl/15 mM NaCl solution (conc in the cytosol) from a 15 mM KCl/150 mM NaCl solution (conc. in blood). Membrane transport_WipaSuginta 1/2556 22 Black lipid membrane (BLM) reconstitution is a technique to study the properties of ion channels Membrane transport_WipaSuginta 1/2556 23 The study of ion channels has been revolutionized by the patch-clamp technique (Erwin Neher and Bert Sakmann,1976) Membrane transport_WipaSuginta 1/2556 24 Acetylcholine receptor is a ligand-gated channels The arrival of a nerve impulse leads to the synchronous export of the contents of some 300 vesicles, which raises the acetylcholine concentration in the cleft from 10 nM to 500 mM in less than a millisecond. The electric organ of Torpedo marmorata, an electric fish, is a choice source of acetylcholine receptors for study. Membrane transport_WipaSuginta 1/2556 25 Binding of acetylcholine opens the Na+/K+ channel The release of acetylcholine causes membrane depolarization at the postsynaptic membrane by increasing the conductance of Na+ and K+. Membrane transport_WipaSuginta 1/2556 26 Active transporters Membrane transport_WipaSuginta 1/2556 27 Primary active transport . Primary active transport, also called direct active transport, directly uses energy to transport molecules across a membrane. Most of the enzymes that perform this type of transport are transmembrane ATPases. Membrane transport_WipaSuginta 1/2556 28 Primary active transport of ions by the Na+/K+ pump in animal cells Membrane transport_WipaSuginta 1/2556 Each time the Na+/K+ pump hydrolyzes one molecule of ATP, three Na+ ions leave the cell and two K+ ions enter it. 29 Secondary active transport In secondary active transport, also known as coupled transport or co-transport, energy is used to transport molecules across a membrane with no direct coupling of ATP, but the electrochemical potential difference created by pumping ions out of the cell is used. Membrane transport_WipaSuginta 1/2556 30 Phloem loading and unloading Membrane transport_WipaSuginta 1/2556 31 Pathways affecting calcium concentrations in muscle cells Membrane transport_WipaSuginta 1/2556 32 Transport of glucose from intestinal lumen into blood stream through basolateral membrane using facilitated passive and active transports Membrane transport_WipaSuginta 1/2556 33