Cellular Neuroscience 207 Ian Parker

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Cellular Neuroscience (207)Ian Parker

• Lecture 2 - Ion channels electrophysiology

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Single ion channels
extracellular
Cell membrane
cytosol
Molecular structure
Physical structure
Functional model
Simplified model
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Single channel kinetics
Transitions from open to shut are
instantaneous Mean open time is a fixed
characteristic of the channel Mean closed time
shortens with increasing stimulus (e.g.
depolarization or agonist concentration) Single
channel current depends on channel
conductance and electrochemical gradient for ion
flow
Channel opening does not require energy
source (ATP), so channel continues to work in
isolated membrane patch. Energy for ion flow
(current) comes from electrochemical gradient
across membrane
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How big are single channel currents?
Amps (log scale)
100 W light bulb
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10-3 (mA)
calculator
10-6 (mA)
action potential at node of Ranvier
Limit of conventional Voltage clamp
e.p.s.c. (current evoked by 1 vesicle of
neurotransmitter)
10-9 (nA)
Single channel currents
10-12 (pA)
One ion per ms
10-15 (fA)
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Single channel current and conductance

• Because single channel current varies with
  membrane potential and ion gradient, a better
  measure is the conductance of the channel (g).
  This is a fixed characteristic (fingerprint) of
  a given channel.
• g i
  / (V-Veq)
• (v membrane potential
  Veq reversal potential for current flow through
  channel)
• Unit of conductance is the Siemen (S 1/Ohm)
  single channel conductances are expressed in pS

Single channel current (pA)
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Line in red shows the current/voltage Relationship
for a single channel. What ion(s) likely pass
through the channel? What is its conductance?
Membrane potential (mV)
100
50
-50
-100
-1
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Range of channel conductances
Conductance (pS)
Maximal conductance of 3 Ao diameter aqueous pore
500
Ca2-activated BK K channel
100
Nicotinic channel
20
K channel in axon
Many channels in 5-30 pS range
Na channel in axon
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Aqueous pore or carrier ? Largest channels
conduct 108 ions per second Fastest enzymes and
transporters have turnover Rates of 105 per sec
(more typically 102-104) So ions transport
must be by diffusion through aqueous pore now
confirmed by structural data.
Limit of present technology
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Store-operated Ca 2 channels
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Recording the activity of single
channelsPatch-clamp technique Neher
Sakmann, 1976, (Nobel Prize1991)
Limitation of voltage-clamp is noise
generated by large area of cell membrane.
Patch-clamp overcomes this by isolating currents
from tiny patch of cell membrane. Sensitive
circuit then amplifies current through channel(s)
in patch, while clamping voltage of pipette
fixed. Current through a single channel is too
small to appreciably alter Resting potential of
cell, so potential across patch Remains constant.
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A commercial patch-clamp amplifier
Patching onto cultured cells under a
microscope
How can you see a channel to know to where to
patch onto the
membrane? You cant! Its a blind fishing
expedition, and takes a lot of patience. Sometimes
you might catch one channel, sometimes many
channels and sometimes nothing. Getting only one
channel is the ideal, as Records with more than
one channel in the patch are hard to
interpret. How do you know if you catch more
than one channel? Sometimes you will see
double openings.
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The giga-seal
Clearly, Rleak must be gtgt Rp for
faithful recording. Rp is pretty much fixed (a
few M Ohm) by the size of the tip (a few
mm). Also, Rleak generates noise from thermal
motion of ions, which decreases as Rleak
increases. So, the higher Rleak can be made, the
better! By using clean cell membrane (e.g.
enzyme treatment to remove connective tissue, or
by using cultured cells) the glass of the
pipette actually sticks to the lipid membrane,
forming a giga-seal (Rleak gt 1 G Ohm) Seal
formation is accomplished by gently pressing the
tip of the patch-pipette against the cell
membrane, then applying gentle Suction.
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Gigaseal recording configurations
An unexpected, but very useful discovery was that
the pipette sticks so tightly after forming
a gigaseal that isolated patches of membrane can
be pulled off intact from a cell.
whole-cell clamp Voltage-clamp of whole cell,
but can be applied to little Cells (e.g.
neurons) that are inacessible to regular
voltage- clamp
cell-attached mode Study single channels in
their intact cellular environment.

Outside-out
excised patch
Study single
channels isolated from cell.
Extracellular face is accessible to Bathing
fluid, so can readily apply neurotransmitters or
other ligands.
Inside-out excised patch. Study single
channels isolated from cell. Cytosolic face is
accessible to bathing fluid, so can readily apply
intracellular second messengers.
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What can patch-clamp recordings tell us?
Obtain long recording with hundreds of events
(openings and closings), then measure
amplitudes, open and closed times for each and
plot distribution histograms
Distribution of single channel amplitudes
Current (i) through a channel is about the same
every time it opens (providing voltage is
constant). However, measurement noise
introduces some variability, so distributions of
channel amplitudes follow a Gaussian with mean
i.
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Mean channel open time is a characteristic of any
particular type of channel, but individual
openings vary randomly
Short opening
Long opening
Random behavior gives rise to exponential
distribution of open times (many short openings,
few long
openings) analogous to radioactive decay
Time constant of decay (t) (time to fall to 1/e
of any Initial value) mean open time
Plotting on logarithmic y-axis transforms
exponential distribution to linear
Exponential distribution of open times on
linear plot
We will talk about distribution of closed times
in a future lecture