Introduction to Membrane Channels and Pumps

1. 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

2. 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.

3. 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?

4. A synapse

7. Ach receptor channel

8. 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.

9. Patch-clamp Technique

10. Patch-clamp Recordings

11. Microinjection of mRNAs for Translation of Ach Receptors In an Oocyte

12. Catalytic Mechanism of Acetylcholine Esterase

13. Opening of the cation selective pore

14. Selectivity of Ach receptor channel

15. 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

16. 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!

18. Depolarization Results in Action Potential

21. Poisons of the Na+ Channel

22. 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?

23. Structure of the Na+ Channel

24. Steric factors and ionic size determine ionic selectivity

25. Neural Transmission

26. 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.

30. 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

31. Active Transport • Two general transport systems • Passive • Active

32. Na+-K+ ATPase Requires Certain Conditions for Activity

33. Na+-K+ Pump Is Oriented on the Plasma Membrane

34. Schematic Representation of the Na+-K+ Pump

35. 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.

36. 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?

38. Mechanism of the Na+-K+ Pump

39. Mechanism of the Na+-K+ Pump

42. Cardiotonic Steroids

43. Digitalis purpurea

44. 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.

45. Sarcoplasmic Reticulum Ca+2 ATPase

46. The cost of transport by Na+-Ca+2 exchanger is paid by the Na+-K+ pump.

47. 3 Classes of Transport