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Introduction to Membrane Channels and Pumps. Ach receptor channel Patch-clamp techniques Ligand-gated and voltage gated channels Action potentials Neurotoxins Molecular structure of sodium channel Gap junctions Common features of membrane channels Active transport
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Introduction to Membrane Channels and Pumps • Ach receptor channel • Patch-clamp techniques • Ligand-gated and voltage gated channels • Action potentials • Neurotoxins • Molecular structure of sodium channel • Gap junctions • Common features of membrane channels • Active transport • Digitalis inhibition of Na-K pump • Ca-ATPase
Membrane Channels and Pumps • Channels: enable ions to flow downhill through membranes • Pumps: use energy (ATP or light) to drive uphill transport. • Ach receptor: nerve cells communicate with other cells by neurotransmitters. • Each vesicle has 104 Ach mols. • After nerve impulse [Ach] increases. • Binding of two Ach mols transiently opens a cation-selective pore. Two mols of Ach should bind to Ach receptor to open channel. Channel opens quickly when Ach increases in synaptic cleft. Stays open one msec. Ach hydrolized quickly.
More About Ach Receptor • Ach binds to postsynaptic membrane, changes its ionic permeabilities, resulting in Na influx and K efflux. • Action potential • Ach receptor channel best understood ligand-gated channel. A ligand –gated channel because can open with a ligand. • Ach receptor 268 kd, 4 kinds of subunits. 2 each has one binding site for Ach. • Monocovalent or divalent cations, not anions, readily pass through Ach channel. • What makes the channel cation-selective?
Patch-clamp Technique • Patch-clamp conductance measurements reveal activities of single channels. • Ion channels best studied by patch-clamp technique (Neher and Sakmann in 1976). • Monitor flow of ions through channels by this technique. • Xenopus oocytes express microinjected mRNAs encoding subunits of Ach receptor.
Action Potentials • Nerve impulse an electric signal produced by flow of ions across plasma membrane of a neuron. • Action potential generated when membrane potential depolarized beyond critical threshold value. • Giant axons of squid used. • Hodgkin and Huxley: “action potentials come from large transient changes in permeability of axon membrane to Na and K’. • Two voltage-gated channels: • Na selective • K selective
Action Potentials Continued • Depolarization of membrane beyond threshold level opens Na channels. • Na’s electrical gradient is high! • Entry of Na depolarizes membrane, more gates open. • + feedback • Action potential very efficient means of signaling over long distances. • Important questions: • How do channels discriminate ions? • Mechanism of voltage-gating? • Mechanism of inactivation? • How do we answer them? By isolating and purifying channels first!
Na Channel • 260 kd • 1 pp chain • 4 repeating units, each has 5 hydrophobic segments, S1, S2, S3, S5, and S6 • Each has one highly +ly charged S4 (Arg and Lys) • S4 is voltage sensor of channel… • Na channel lets Na pass 11X more than K. Why? • How is selectivity achieved? • K channels highly selective also. Why?
Gap Junctions • Gap junctions important intercellular communications. • Sugars, amino acids, nucleotides flow through gap junctions but proteins, polysaccharides, nucleic acids too large. • Gap junctions: • Heart • Bone • Lens • Structure of gap junctions: • Connexin (32 kd transmembrane protein) • 6 of them form connexon • 2 connexon connexin (channel) • Gap junctions differ from other channels: • Traverse two membranes, connect cytosol to cytosol, synthesized by different cells. • Open: seconds to minutes; Close: Ca2+ or H+ increase.
Membrane Channels Have Common Features • Formed by 4-6 identical units • Pore lies along symmetry axis of channel • Side chains inside channel make them selective • Diameter of narrowest part of pore helps selectivity • Channels are allosteric proteins controlled by membrane potential, modulators, or covalent modifications
Active Transport • Two general transport systems • Passive • Active
Na+-K+ Pump • Three Na and two K transported per ATP hydrolysis (in 10 ms). • Important feature of pump is that ATP not hydrolyzed unless Na and K transported. • How do phosphorylation and dephosphorylation of ATPase lead to transport of Na and K across membrane? • 3D structure of pump not known in detail. • Proposed model by Oleg Jardetzky: • Must contain large cavity to hold small molecule. • Must have at least two conformations. • Must have different affinities.
Na+-K+ Pump Continued • ATP hydrolysis drives pumping of Na and K ions across plasma membrane. • Four important facts about pump… • Structure of pump… • How does ATP drive active transport of Na and K?
Digitalis • Digitalis inhibits Na-K pump by blocking dephosphorylation. • Digitalis, steriod derived from plants. • Ouabain is one used to treat heart failure. • Inhibit dephosphorylation of E2-P when applied on extracellular face of membrane. • Na gradient decreases Ca2+ increases in cell • Na-Ca exchanger in plasma membrane uses electrochemical gradient of Na to pump Ca out of cell. • 3 Na enter for each Ca that exits. • Cost of transport by exchanger paid by Na-K pump.