Lecture 5 Channels Patch clamp and sequence analysis

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

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

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

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

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

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

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

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

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

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

Properties of channels n I = 2.7pA  ions/s  ions/ms Number of obs

Properties of channels nRate of opening & closing is very fast

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

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

Summary so far nIn a small patch, hold V fixed and measure I nsize is pS

Multiple channels? nEmbryonic rat ACh channels (cell attached)

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

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

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

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

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

Simulation nSimulate Na + channel u Bezanilla Bezanilla

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

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

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

Phylogeny Na Ca K

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

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

Mutation of Na channel nChange I to V at 1160

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