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1. Action Potential

2. Ionic Basis of Action Potential

Action Potentials

• What are they?– Rapid reversal of the resting membrane

potential

cell

0 mV

-80 mV

3 ms

Electrophysiology Techniques

Lolligo pealeii

Squid giant axon-1 mm in diameter-1000X larger than most

axon

Recording electrode

Reference electrode

stimulus

Action Potentials

Important concepts:• Threshold• All or none• Regenerative• Conduction along axons

Action Potential

0 mV

-80 mV

Rising phase ordepolarization

Falling phase orRepolarization

Resting membranepotential

Threshold Potential

Undershoot or after-hyperpolarization

Overshoot

Reminders…

1. Action potential is a rapid reversal of Vm

2. Vm is dominated by the equilibrium potential of the most permeable ion

3. Permeability controlled by ion channels

Ionic Basis of Action Potentials

0 mV

-80 mV

PK>>PNa

PNa>>PK

PK>>PNa

ENa

EK

Time

K+

Na+ Voltage-gated channels

K+ leak channel

Na+

K+

Section of Squid Axon

Me

mbr

an

e P

ote

ntia

l

time

K+

Na+

Na+

K+

time

Me

mbr

an

e P

ote

ntia

l

1. At rest only K+ leak channels open, PK>>PNa

2. With stimulus, voltage-gated Na channels open, PNa>>PK

Na+ flows into the cell carrying positive charge

3. Delayed opening of voltage-gated K channels, PK>>PNa

K+ flows out of cell removing positive charge

K+

Na+

Na+

K+

How do we know Na+ important for depolarization?

0 mV

-80 mV

Replace Na+ in extracellular bath with impermeable

cation - cholineNormal

Low Sodium

Ion currents underlying the AP

• Use voltage-clamp technique to measure currents

• Measure currents in the presence and absence of Na+

• What are ionic currents?– So far, voltage (V)– When ions move current (I)– Movement through channel is resistance (R)

• Reciprocal is conductance (g)

Ohm’s law

I=V/R or I=gV

Where g = 1 / R

• More properly

Iion = gion X emfion

• Iion is ionic current

• gion is ionic conductance

• emfion is the electromotive force acting on an ion

• emfion = Vm - Eion

Total membrane potential Nernst potential for the ion

Therefore,

• gion is controlled by ion channels

• If all channels closed, g = 0 and no ions flow

• if Vm = Eion then emf = 0, and no ions flow

( )ion ion ionI g Vm E

• How are ion currents measured?

• Voltage-clamp

Electrophysiology TechniquesVoltage clamp

Squid axon

Recording electrode

Reference electrode

Command Signal

ImCurrent output

Control amplifier

Voltage output

+ -

Currentelectrodes

Membrane currents

• Measure ionic currents from squid axon– To determine contribution of Na+ and K+

Measure in normal saline with Na+ and Na+-free saline

Ion currents underlying the AP

Membrane

Potential

Ionic

Currents

Total current normal saline

K+ current Na+ free saline

outward

inward

Ion currents underlying the AP

Ionic

Currents

Na+ current

outward

inward

Ionic

Currents

Total current normal saline

K+ current Na+ free saline

outward

inward

Subtract K+ current from total

Ion currents underlying the AP

1. The Na+ current activates quickly and then inactivates quickly

2. The K+ current activates more slowly and persists longer

Ionic Basis of Action Potentials

0 mV

-80 mV

PK>>PNa

PNa>>PK

PK>>PNa

ENa

EK

Time

Ion currents underlying the AP

Membrane

Potential

Ionic

CurrentsK+ current

After-hyperpolarization

Pk(leak) + Pk(volt)Pk(leak)

• The after hyperpolarization coincides – with the persistent K+ current and absent Na+

current

Stimulus & Threshold

• The stimulus depolarizes the membrane– Experimentally applied current– Synaptic potential– Receptor potential

Threshold

• The membrane potential at which

Na flowing into the cell exactly equals the

K flowing out of the cell

• A fraction more stimulus depolarization is required to ‘fire’ an action potential

Threshold Potential

0 mV

-80 mV Small stimulus

Below threshold

Larger stimulus

Reaches threshold

Membrane

depolarization

Increased Na

permeabilityNa+ entry

Positive Feedback

The AP is regenerative and

displays all-or-none behaviour

Why does the AP stop rising?

1. As VmENa, Na+ inflow stops

2. Na+ channels

inactivate

3. K+ channels open, K+ outflow starts

ENa

Refractory Period

1. A second stimulus very soon after the first will not fire an AP (Absolute)

2. With a delay, a second stronger stimulus will cause a small AP (Relative)

3. With longer delay a second AP can be fired

Absolute refractory

period

Relative refractory

period

A

B

C

Why is there Refractory Period?

• The Na channel stays inactivated for a short period of time after it closes

InactivatedOpenClosed

Active

Closed

Active

Summary & Key Concepts

1. The AP is controlled by rapid changes in ionic permeability

2. Permeability is a function of voltage-gated ion channels

3. Threshold potential

4. Positive feedback

5. Refractory period has two phases