Transcript Cellular Neuroscience (207) Ian Parker

Cellular Neuroscience (207)
Ian Parker
Lecture # 4 - Ion channels:
electrophysiology
http://parkerlab.bio.uci.edu
Single ion channels
extracellular
Cell
membrane
cytosol
Molecular structure
Physical structure
Functional model
Simplified model
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
How big are single channel currents?
Amps (log scale)
Limit of conventional
Voltage clamp
1
100 W light bulb
10-3 (mA)
calculator
10-6 (mA)
action potential at node
of Ranvier
10-9 (nA)
e.p.s.c. (current evoked by 1 vesicle
of neurotransmitter)
10-12 (pA)
Single channel currents
10-15 (fA)
One ion per ms
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)
1
Line in red shows the current/voltage
Relationship for a single channel.
What ion(s) likely pass through the channel?
Membrane potential (mV)
-100
-50
50
100
What is its conductance?
-1
Range of channel conductances
Conductance (pS)
500
Maximal conductance of 3 Ao diameter
aqueous pore
Ca2+-activated ‘BK’ K+ channel
100
Nicotinic channel
20
K+ channel in axon
Many channels in
5-30 pS range
Na+ channel in axon
10
Aqueous pore or carrier ?
Limit of present
technology
Largest channels conduct 108 ions per second
1
Store-operated
Ca 2+ channels
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.
Recording the activity of single channels
‘Patch-clamp’ technique : Neher & Sakmann, 1976,
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.
(Nobel Prize1991)
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 can’t! It’s 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.
The ‘giga-seal’
Clearly, Rleak must be >> 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 > 1 G Ohm)
Seal formation is accomplished by gently
pressing the tip of the patch-pipette against
the cell membrane, then applying gentle
Suction.
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.
‘cell-attached’ mode
Study single channels
in their intact cellular
environment.
‘Inside-out’ excised
patch.
Study single channels
isolated from cell. Cytosolic face is accessible
to bathing fluid, so can readily apply intracellular
second messengers.
‘whole-cell clamp’
Voltage-clamp of
whole cell, but can
be applied to little
Cells (e.g. neurons)
that are inacessible
to regular voltageclamp
‘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.
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.
Mean channel open time is a characteristic of any particular type of channel,
but individual openings vary randomly
Long opening
Short opening
Random behavior gives rise to exponential distribution of open times (many short openings, few
long openings) : analogous to radioactive decay
Exponential distribution
of open times on linear
plot
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
[We will talk about distribution of closed times in a future lecture]