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