1. Lecture 5 Channels Patch clamp and sequence analysis

2. Aims nTo know about the patch clamp method nto know about diversity of channels ncompare with Dale Sanders module 610 Membranes lecture!

3. Reading matter nBooks: u Levitan & Kaczmarek "The Neuron" (2001, 3rd ed) OUP u Purves, D (et al) (2001) Neuroscience Sinauer

4. Papers: u Sakmann B, Bormann J, Hamill OP. Ion transport by single receptor channels. Cold Spring Harb Symp Quant Biol. 1983;48:247-257 u Neher E, Sakmann B. The patch clamp technique. Sci Am. 1992 Mar;266(3):44-51 u Catterall WA From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-Gated Sodium Channels Neuron 2000 26: 13-25

5. Revision nResting and action potentials nSynaptic receptors nHow do we know ?

6. Cell-attached nGlass pipette u filled with saline u coated with sylgard ncurrent amplifier nGigaOhm seal u implies the distance from glass to membrane is about the same as a chemical bond

7. Whole Cell nCell-attached + suck u breaks membrane u effective voltage clamp of small cells

8. Whole cell allows… nexchange of pipette solution with cell, so introduce u Dye F Lucifer yellow u transduction reagents LY

9. Outside Out nStart with whole cell nPull away, neck breaks off nGives access to extracellular surface, with intracellular surface controlled

10. Inside - Out nStart with cell-attached, and pull away nExtracellular surface is inside the pipette, intracellular surface can be manipulated

11. Properties of channels nChannels have a fixed size Number of obs ACh in cell-attached pipette

12. Properties of channels n I = 2.7pA  1.6 10 7 ions/s  1.6 10 4 ions/ms Number of obs

13. Properties of channels nRate of opening & closing is very fast

14. Channels & Ohm’s Law nV = IR  I = V/R ng is conductance nI = V g  g = I/V u g is measured in mho or Siemens

15. Channels & Ohm’s Law nhigh current nStraight line of Ohm’s law MEAN nions don’t interact with channel pore u not a carrier u not a pump u just a hole

16. Summary so far nIn a small patch, hold V fixed and measure I nsize is 4 - 200 pS

17. Multiple channels? nEmbryonic rat ACh channels (cell attached)

18. Opening and closing nLigand gated channels u Openings in bursts n exponential decline u each opening event is random (independent) Open 10.6ms Closed 18 ms

19. Opening and closing open closed As [ACh] increases the binding of 2 Ach becomes more likely

20. Opening and closing nBursts of opening u 2ACh + R  ACh + R-ACh  2 R-ACh  2 R-ACh* u multiple openings while ACh is bound

21. Opening and closing nVoltage gated e.g. K + channel u opening is more likely the more the membrane is depolarised

22.   Opening and closing nSodium channel u 3 models of closed / open / inactivated

23. Simulation nSimulate Na + channel u Bezanilla Bezanilla

24. Opening and closing nMany channels need to be phosphorylated to open u Ca 2+ channel u opens to + step u wash out - stays shut u PKA restores

25. Summary so far nIn a small patch, hold V fixed and measure I nsize is 4 - 200 pS nLigand gated channels nVoltage gated channels nmodulated by internal state

26. Sequence of channel nChannels have subunits u Na+ monomer, u K+ tetramer  -helix in membrane u 6 spans /subunit u homology! Charged helix makes pore

27. Phylogeny Na Ca K

28. Channel subtypes nE.g. Ca channel u L-type 25pS, - 10mV u P-Type 25pS, - 10mV u T-type 8pS, -40mV n L-type sensitive to dihydropyridines u nifedipine u nitrendipine n block opening of channel n important as relaxants of blood vessels in angina, hypertension

29. Mutation of Ca ++ channel  disease Migraine ataxia night blindness paralysis

30. Mutation of Na channel nChange I to V at 1160

31. Summary to end nIn a small patch, hold V fixed and measure I nsize is 4 - 200 pS nLigand gated channels nVoltage gated channels nmodulated by internal state nmany sub-types nmutation can lead to disease