Transcript Voltage clamp experiment

The two ions that make up the action potential
The clamp half of the voltage clamp
Axial wire electrodes used in Voltage-Clamp
experiments
• Hodgkin and Huxley set out to determine which ions
carry the current and how the underlying membrane
permeability mechanisms worked.
• They reasoned that each ion seamed to move
passively down their its electrochemical gradients.
• Currents carried by Na+ should move inward at
potentials negative to the equilibrium
potential, ENa and outward for potentials
positive to Ena
• If the membrane is clamped at ENa, Na+ ions should
make no contribution to the observed membrane
current, and if the current reverses sign around ENa, it
is probably carried by Na+ ions.
• Similar assumptions were made for K+, Ca2+ and Cl-.
• Finally, ions could be or removed from the external
solution. added or removed
At resting conditions
• The steady state membrane potential will be
between ENa and EK, a voltage at which the
inward sodium current exactly balances the
outward potassium current
• The total membrane current is zero
( INa + IK = 0).
If the steady state is perturbed
• If pNa is increased, there will be an increase in
INa .
• This sodium influx causes Em to move in a
positive direction from its original value.
• With depolarization potassium current
increases because of the difference between
Em and EK.
New Steady State
• The membrane will reach a new steady-state,
governed by the new ratio pNa/pK .
• Both INa and IK are larger than they were
initially.
• The two currents will again be equal to each
other.
• Illustrated in the next slide.
• If Em Eclamp are equal there will be no current
injected in to the cell.
• If at time = t1 we turn on the voltage clamp
and there is a sudden increase in sodium
permeability. The voltage clamp will
immediately detect this change, the voltage
clamp amplifier will inject negative current
into the axon.
• In current terms
•
INa = gNa (Em – ENa)
• or solving for gNa and rearranging:
•
gNa = INa/(Em – ENa)
• here Em is the voltage set experimenter as the
command voltage and ENa is the Nernst equilibrium
potential for sodium.
• Here we have talked in terms of permeability but
measured in terms of conductance. They are not the
same, only their time course may be the same
The voltage clamp adds and additional artificial
source of current
Voltage camp value placed at ENa
Method for showing the sodium component by varying the
sodium concentration using choline chloride for NaCl.
A more realistic rendition of voltage clamp
curve.
Clamped voltages (from Hillel)
Another drawing showing 5 clamped voltages
and their respective analog conductances
• Note in the previous slide, (b) curve 2 dips
deeper down than curve 1, Why?
Conductance curves for Na and K
Using specific agents to block Na+ or K+ channels
in voltage clamp studies
Plotted vs. experimental derived data points
• In the next slide, are conductance curves of H
& H data.
• The circles are data points derived from
experimental results, the curve is drawn
according to the equations used for
calculating conductances.
Conductance curves for Na and K
Action Potential vs conductance and
local currents
Logical extension of voltage clamp experiments
• A reductionist view would ask what would the
current be for hydrophilic single channels.
• Neher and Sakmann developed the patchclamp
A glass pipette is pulled so that the open
diameter is around 1 micron
• The glass is fire-polished so there are no ragged
edges.
• The pipette is back filled with an ion solution; NaCl
or KCl at around 3M.
• When applied to the membrane, a small suction pulls
the membrane into the pipette with one or more
channels. This results in a resistance of over a gigasill
ohms (see next slide)
Patch clamp
General characteristics of patch-clamp data
• For all patch-clamp data, abrupt transition
state have been recorded.
• Current flows during these open states, but no
current flows in the closed states.
• The preceding slide show these changes and
computer idealized rendition of these
changes.
Types of recording techniquws
• Note that the gigaseal of the membrane –
pipette connection is both electrically tight
and machanically tight.
Ion flow through channels is fast.
• It should be noted that one is observing the
mechanical changes that are operating within a
rather large protein complex structure.
• A typical chemical enzyme assay in a test tube
measure the activity of 1010 or more protein
molecules.
• Modern electrical devices (current to voltage
converters) can measure as little as 10-13A (0.1pA) of
current.
• The above would not be possible unless the current
flow was fast.
What the data allows one to assume about the ion
channel
• Typical values are 1-20 pA range.
• This translate to the movement of 0.6-12 x 107
(10,000,000) ions per second through the channel.
• The only metabolic turn over rate this fast can be via
diffusion through a pore.
• The ion channel with this fast of a rate allow the
assumption that the pore must be a hydrophilic lined
channels that works by diffusion.
Answers sought by patch – clamp studies
• Single channel conductance: a measure of the rate which ions
pass through a channel.
• Ion selectivity: the nature of the ions that are allowed to p ass
through a one channel.
• Gating: the opening and closing of a channel unrer the
influence of such factors as the transmitter - membrane
voltage, the binding of neurotransmitters, hormones, and
other agents to sites on the outside of the channel, and the
actions of certain intracellular metabolites and enzymes.
• Pharmacology: the susceptibility of the channel to various
compounds that may block the pore or otherwise influence
channel properties.
Single channel conductance
• As in voltage clamp measure, the voltage across the
membrane can be set by the investigator.
• The size of the current that flows across the
membrane can be plotted against voltage (see next
slide).
• For many channels a straight line is obtained over a
wide range of voltages.
• Two pieces of information are gathered; unitary
conductance (b, slide below)and the reversal
potential (c, slide below).
Single Channel Conductance (g = ∆I/∆V)
• Recalling Ohms law
•
E = RI or E/R =
•
1/R = I/V
1/R = g = ∆I/∆V
Single channel conductance is in the order of
picosiemens or 10-12 S.
The range of measured currents todate has be from 5400 pS.
Ion selectivity
• One of the main features is the ability to test for the
lack of current when the membrane is at t he
equilibrium potential for that ion.
• In c, two slides back the roll over from the straight
line is an example. The channel may still open or
close, but no current runs through the channel.
• The structure of the pore is determinant as to which
ion goes through the channel.
• Ions, together with their associated water cloud
Ion selectivity (cont.)
• Must make a tight fit with the most narrow
point in the channel.
• This means the channel is a selective filter.
• Evolutionary pressures have designed the
channels to just fit the size of neuron
channels.
Gating
• Ion channels a dynamic structures, they are open,
open but inactivated or closed.
• This is readily apparent from reading the patch clamp
charts of single.
• The amount of time spent in either of the two states,
open/closed, will depend on the relative values of
free energy.
• The free energy is reflected by easily measured
quantities, the rate constants for channel opening
and closing
Gating states
Gating in patch clamp
Voltage-dependent channels
• When one says the gate is open or closed, we mean
that the relative free energies between open and
closed states have changed so that the channel is
more likely to be open or closed than it was
previously (b, slide below).
• The next slide is representative of channel opening
and closing for a potassium voltage-gated channel at
different clamped voltages.
• As the potential is made more positive the activation
increases such that at +20 mV or higher the channel
is totally activated
Voltage-gated channels (cont)
• Note the time the channel spends in the open and closed
state.
• Further, note that the amplitude of the open state changes,
which reflects the fact that the channel is sensitive to the
driving state of the diffusion.
• The curve is sigmoid, the steepness of which reflects the
channels sensitivity to voltage.
• The change in conductance at the top of the curve reflects the
change in current flowing through the channel. This is called
rectification and reflects that the membrane changes
resistance as a function of voltage( this is not voltagedependent gating)
m Gates sensitive voltage
N Gates sensitive to voltage
• The whole cell sodium current is the sum of the
currents passing through all of the sodium channels
in the plasma membrane.
• or,
•
I = Npoi
• where is macroscopic current, N is the number of
channel, po is the probability that any channel is
open and i is the current travleing through a single
channel.
Macroscopic Ion currents Result from Activity of
Populations of Ion Channels
Unitary K channels
Unitary Na channels