Download Electrophysiology membrane potential

2005-06
Electrophysiology
Membrane potential (Kandel Ch 7 – pp 125)
Information within and between neurons: by
electrical and chemical signals.
Receptor, synaptic and action potentials across the
membrane:
a. Transient electrical signals that carry time- and
intensity-sensitive information (how qualitysensitive information is transferred?).
b. Produced by changes in the current flow into and
out of the cell.
c. Current is controlled by ion channels in the
membrane.
Ion channels:
a. Resting – normally open and maintain the resting
membrane potential.
b. Gated – closed during resting potential and
opening regulated by changes in:
* Membrane potential - voltage dependant,
* Ligand binding - associated with receptor,
* Membrane stretch – sensory receptor.
Resting membrane potential:
a. Due to separation of charges across the membrane.
b. Vm = Vin – Vout = ~ -60 mV
c. Not a stable condition since frequent opening of
ion channels.
Electrical current flows into and out of the cell:
a. Carried by ions (positive cations and negative
anions). Normal current in socket is carried by
electrons.
b. Direction of current is defined as direction of net
movement of positive ions.
c. Current in = depolarization;
Current out = hyperpolarization.
Changes in membrane potential:
a. Electrotonic (graded) – passive changes in
membrane potential if not leading to opening of
gated channels.
b. Electrotonic – hyperpolarizations and small
depolarizations.
c. Action potentials – opening of voltage sensitive
channels at certain voltage threshold.
Resting membrane potential:
a. Asymmetric distribution of ion across the
membrane
b. Out – more Na+ and Clc. In – more K+ and organic anions ANernst equation:
a. Used to find the equilibrium potential of any ion
present on both sides of the membrane permeable
to that ion.
b. K+ = -75 mV
c. Na+ = +55 mV
d. Cl- = -60 mV
e. A- = membrane not permeable
Ion Equilibrium Potential =
(gas constant * temp) / (ion valence * Faraday constant)
* ln (ion concentration out / ion concentration in) =
Ion constant * ln (ion concentration out / ion
concentration in)
Equilibrium potential:
a. The electrical force related to potential gradient
equals the chemical force related to concentration
gradient.
b. Ion flux = (electrical force + chemical force) *
membrane conductance.
Resting channels – resting potential:
a. Electrophysiology and flux studies using
radioactive tracers.
b. Nerve cells at rest are permeable to Na (low
amount of resting channels), Cl, and K (high
amount). Implication for resting potential ???
c. Membrane is not permeable to A anions.
d. Fig. 7-4 (pp 130).
e. Resting potential affected by K equilibrium
potential because of the many resting channels
(-75mV).
f. Resting potential is slightly depolarized from the K
equilibrium potential due to influx of Na. But only
slightly because not too many resting Na channels.
g. Influx of Na to the cell reduces the electrical
inward force on K and therefore K starts to flow
out, thus compensating for the Na influx, but at
new resting potential (-60 instead of -75mV).
Wilson & Kawaguchi, 1996 (J. Neurosci., 16: 2397-2410)
Active pumping of the ions:
a. The outflow of K and the inflow of Na is balanced
at the new resting potential.
b. This steady leak of ions across the membrane has
to be compensated to prevent decrease of the
concentration gradients.
c. This is achieved by the Na-K active pump using
ATP derived energy.
d. Thus, at the resting potential the cell is not at
equilibrium (there is ion flow) but rather in a
steady state: continuous ion flow counterbalanced
by Na-K pump (Na/K = 3/2, hyperpolarization
effect).
e. The pump has binding sites for Na and ATP on the
intra-cellular surface of the membrane and for K
on the extra-cellular surface.
f. The resting potential is just about the equilibrium
potential of the Cl and therefore it has no effect on
the resting potential.
Action potential:
a. Once the membrane potential exceeds some
threshold value, voltage gated Na channels open
rapidly.
b. The result is depolarization toward the Na
equilibrium potential +55mV.
c. The potential does not reach +55 mV since there is
constant efflux of K and influx of Cl.
d. This mechanism of spike is terminated by gradual
inactivation of the voltage gated Na channels and
by opening of voltage gated K channels. This
creates a net efflux of positive charges from the
cell until the cell reaches back the resting potential.
The action potential (Kandel Ch 9 – pp 150)
Action potential
a. Action potential is generated and propagated by
voltage-gated ion channels. It is an active signal
conductance over long distance without attenuation of
amplitude (unlike the passive current).
b. Ion conductance across the membrane increases
during the action potential.
c. Na influx is responsible for the rising phase of the
action potential due to a transient increase in
membrane permeability to Na. What is the effect of
increase Na concentration?
d. K efflux is responsible for the falling phase of the
action potential due to a transient increase in
membrane permeability to K. What is the effect of the
increase in K concentration?
How the properties of the voltage-gated Na and K
channels are studied?
The intention was to vary the voltage across the
membrane and observe the changes in Na and K
conductance.
a. Before the voltage clamp technique was developed,
there was a problem to stabilize the membrane
potential because of a positive feedback:
experimental depolarization opens the Na voltagegated channels which lead to inward flux of Na
which cause depolarization and so on, until the
membrane reaches the peak of the action potential.
b. The voltage clamp technique prevents the opening
and closing of the voltage-gated ion channels upon
change of the membrane potential. It does so by
injecting into the cell a current that is equal and
opposite to the current flowing through the voltagegated channels.
c. The current that has to be injected to keep the
membrane potential stable is a direct measure of the
ionic current through the ionic channels.
Voltage clamp technique for studying membrane currents of a
squid axon.
The device permits the simultaneous measurement of the current
needed to keep the cell at a given voltage (4). Therefore, the
voltage clamp technique can indicate how membrane potential
influences ionic current flow across the membrane. This
information gave Hodgkin and Huxley the key insights that led
to their model for action potential generation.
Experiment with voltage clamp technique (Fig 9-3,
p:153; Box 9-1, p.152)
a. Experiment starts at resting potential.
b. 10mV depolarization pulse through the voltage
clamp electrode leads to transient outward
capacitance current followed by a leakage current.
Capacitance current (related to the lipid layer) is
starting only with a change in the membrane
potential. It is instantaneous as it discharges/charges
through the resistance of the membrane. It is much
faster than the dynamics of the ionic currents.
Leakage current is mainly through the resting ion
channels, mainly the K channels (highest
permeability), but also Na and Cl resting channels.
c. 20 mV depolarization pulse leads to larger currents.
First the outward capacitance discharge current
followed by outward leakage current. Then inward
and then outward current, most likely due to
conductance through Na and K voltage gated
channels.
d. To separate the time course of the last two currents,
the axon is treated with selective blockers of the Na
or K channels.
e. Such experiments demonstrated simultaneous fast
opening of the Na channels and slower opening of
the K channels.
f. Such experiments are then repeated at different
voltage depolarization steps.
Refinement of the voltage clamp technique is the
patch clamp technique for recording in single ion
channels (Box 6-1, p.111).
http://www.science-display.com/patchclamp.html
The pipette electrode is filled with a ligand (for
example) that activates the channel to record a
transmitter-gate channels in the membrane.
Four configurations in patch clamp measurements of ionic currents.
Back to spike mechanisms:
Fig 9-6 and Fig 9-7 (p 156) shows that depolarization
pulses cause fast increases in conductance of Na voltage
dependent channels (inward current) which are
inactivated even if the depolarizing pulses are still on.
The same depolarizing pulses are slowly increasing the
conductance of voltage dependent K channels which stay
open as long as the pulses are on.
The above sequence of Na and K channels opening
define the shape and the duration of the action potential.
A small riddle is why the action potential is all or none
phenomena inspite of the fact that the voltage dependent
Na channels are opened already with subthreshold
depolarizations. The answer is that until certain
depolarization level the inward Na is opposed by
outward K and Cl currents. However at certain level of
depolarization the Na current is stronger than the other
currents because of the great sensitivity of Na channels
to voltage and its fast opening dynamics. At this point
the net current of Na is stronger than the other currents
and produces a regenerative depolarization.
Life after the spike:
Fig 9-9 shows the complex votage dependent dynamics
of the Na channel. It has two gates: activation gate and
inactivation gate. Only at the depolarization level the two
gates are open and therefore produce the rising phase of
the spike.
As the inactivation Na gate is very slow to open, it
causes an absolute refractory period after the spike
termination. During this period no depolarization current
can produce a new spike.
Residual opening of the voltage dependent K channels
after the spike cause the relative refractory period during
which stronger depolarization is needed to produce a
spike (see Fig 9-6 for the residual opening of the K
channels after the depolarizing current is already off).
The story of the Ca2+:
Unlike other ions, the concentration of Ca which is
initially very low in the cell, is increasing significantly
during depolarization due to the opening of voltage-gated
Ca channels. The effects of the inward flow of Ca are
limited since Ca increases the opening of the K channels
(efflux of K) and closing of the Ca channels by the inside
Ca concentration. Eventually the influx of Ca serves as a
second messenger for changing the properties of some
channels.
Excitability at different regions of the neuron:
In some neurons the dendrites have voltage-gated ion
channels, such as Ca, K and also Na. activation of these
channels modify the passive, electrotonic conduction of
synaptic potentials. In some of these neurons the spike
can be propagated from the axon hillock (the integration
zone due to highest concentration of voltage gated Na
channels) back into the dendrites. This kind of
mechanism was shown recently to be involved in
learning phenomena and explains how certain region of
the dendrite may be potentiated.
Excitability properties vary among neurons
How the neuron responds to synaptic input is determined
by the proportions of different types of voltage-gated
channels in the neuron's integrative and trigger (integrate
and fire) zone. Some cells respond with a single spike
others with burst of spikes. Various response options are
shown in Fig 9-11. Particularly interesting is section B
and C. Section B shows the generation of delayed burst
activity after prolonged inhibition which causes the
voltage gated Ca channel to recover from inhibition.
Section C shows repeated bursting (contribution to
normal and epileptic EEG rhythmic patterns). This
pattern is also caused by strong effects of Ca channel
activated during Na influx and closed between the bursts,
thus allowing hyperpolarization.
END