Lecture 5 Channels Patch clamp and sequence analysis

Lecture 5 Channels

Patch clamp and sequence analysis

Aims

To know about the patch clamp method to know about diversity of channels compare with Dale Sanders module 610

Membranes lecture!

Reading matter Books:

Levitan & Kaczmarek "The Neuron" (2001, 3rd ed) OUP

Purves, D (et al) (2001) Neuroscience Sinauer

Papers:

Sakmann B, Bormann J, Hamill OP. Ion transport by single receptor channels. Cold Spring Harb Symp Quant Biol. 1983;48:247-257

Neher E, Sakmann B. The patch clamp technique. Sci Am. 1992 Mar;266(3):44-51

Catterall WA From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-Gated Sodium Channels Neuron 2000 26: 13-25

Revision

Resting and action potentials Synaptic receptors

How do we know ?

Cell-attached

Glass pipette filled with saline coated with sylgard

current amplifier GigaOhm seal

implies the distance from glass to membrane is about the same as a chemical bond

Whole Cell

Cell-attached + suck breaks membrane effective voltage

clamp of small cells

Whole cell allows…

exchange of pipette solution with cell, so introduce Dye

Lucifer yellow transduction

reagents

LY

Outside Out

Start with whole cell Pull away, neck breaks off Gives access to extracellular surface,

with intracellular surface controlled

Inside - Out

Start with cell-attached, and pull away

Extracellular surface is inside the pipette, intracellular surface can be manipulated

Properties of channels Channels have a

fixed size

Number of obs

ACh in cell-attached pipette

Properties of channels

I = 2.7pA 1.6 •107 ions/s 1.6 •104 ions/ms

Number of obs

Properties of channels Rate of

opening & closing is very fast

Channels & Ohm’s Law V = IR I = V/R g is conductance

I = V g g = I/V g is measured

in mho or Siemens

pSV

Ag 40

1050

1023

9

Channels & Ohm’s Law high current Straight line of

Ohm’s lawMEAN ions don’t

interact with channel pore not a carrier not a pump just a hole

Summary so far

In a small patch, hold V fixed and measure I

size is 4 - 200 pS

Multiple channels?

Embryonic rat ACh channels (cell attached)

Opening and closing

Ligand gated channels Openings in bursts

exponential decline each opening event

is random (independent)

Open10.6ms

Closed18 ms

Opening and closing

open

closed

As [ACh] increases the binding of 2 Ach becomes more likely

Opening and closing

Bursts of opening 2ACh + R

ACh + R-ACh 2 R-ACh 2 R-ACh*

multiple openings while ACh is bound

Opening and closing

Voltage gated e.g. K+ channel opening is more likely the more the

membrane is depolarised

Opening and closing

Sodium channel 3 models of closed / open / inactivated

Simulation

Simulate Na+ channel Bezanilla

Opening and closing

Many channels need to be phosphorylated to open Ca2+ channel opens to +

step wash out -

stays shut PKA restores

Summary so far

In a small patch, hold V fixed and measure I

size is 4 - 200 pS Ligand gated channels Voltage gated channels modulated by internal state

Sequence of channel

Channels have subunits Na+ monomer, K+ tetramer

-helix in membrane 6 spans /subunit homology!

Charged helix makes pore

Phylogeny

Na

Ca

K

Channel subtypes

E.g. Ca channel L-type 25pS, -

10mV P-Type 25pS, -

10mV T-type 8pS,

-40mV

L-type sensitive to dihydropyridines nifedipine nitrendipine

block opening of channel important as relaxants of

blood vessels in angina, hypertension

Mutation of Ca++ channel disease

Migraineataxianight blindnessparalysis

Mutation of Na channel Change I to V at 1160

Summary to end

In a small patch, hold V fixed and measure I

size is 4 - 200 pS Ligand gated channels Voltage gated channels modulated by internal state many sub-types mutation can lead to disease