Lecture 5 Channels Patch clamp and sequence analysis.
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Transcript of Lecture 5 Channels Patch clamp and sequence analysis.
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Lecture 5 Channels
Patch clamp and sequence analysis
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Aims
To know about the patch clamp method to know about diversity of channels compare with Dale Sanders module 610
Membranes lecture!
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Reading matter Books:
Levitan & Kaczmarek "The Neuron" (2001, 3rd ed) OUP
Purves, D (et al) (2001) Neuroscience Sinauer
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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
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Revision
Resting and action potentials Synaptic receptors
How do we know ?
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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
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Whole Cell
Cell-attached + suck breaks membrane effective voltage
clamp of small cells
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Whole cell allows…
exchange of pipette solution with cell, so introduce Dye
Lucifer yellow transduction
reagents
LY
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Outside Out
Start with whole cell Pull away, neck breaks off Gives access to extracellular surface,
with intracellular surface controlled
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Inside - Out
Start with cell-attached, and pull away
Extracellular surface is inside the pipette, intracellular surface can be manipulated
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Properties of channels Channels have a
fixed size
Number of obs
ACh in cell-attached pipette
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Properties of channels
I = 2.7pA 1.6 •107 ions/s 1.6 •104 ions/ms
Number of obs
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Properties of channels Rate of
opening & closing is very fast
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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
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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
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Summary so far
In a small patch, hold V fixed and measure I
size is 4 - 200 pS
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Multiple channels?
Embryonic rat ACh channels (cell attached)
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Opening and closing
Ligand gated channels Openings in bursts
exponential decline each opening event
is random (independent)
Open10.6ms
Closed18 ms
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Opening and closing
open
closed
As [ACh] increases the binding of 2 Ach becomes more likely
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Opening and closing
Bursts of opening 2ACh + R
ACh + R-ACh 2 R-ACh 2 R-ACh*
multiple openings while ACh is bound
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Opening and closing
Voltage gated e.g. K+ channel opening is more likely the more the
membrane is depolarised
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Opening and closing
Sodium channel 3 models of closed / open / inactivated
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Simulation
Simulate Na+ channel Bezanilla
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Opening and closing
Many channels need to be phosphorylated to open Ca2+ channel opens to +
step wash out -
stays shut PKA restores
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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
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Sequence of channel
Channels have subunits Na+ monomer, K+ tetramer
-helix in membrane 6 spans /subunit homology!
Charged helix makes pore
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Phylogeny
Na
Ca
K
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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
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Mutation of Ca++ channel disease
Migraineataxianight blindnessparalysis
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Mutation of Na channel Change I to V at 1160
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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