LECTURE 6: ACTION POTENTIAL INITIATION AND PROPAGATION

REQUIRED READING: Kandel text, Chapter 9

Action potential (AP) is a brief spike of strong membrane depolarization at a point along the axon caused by inward current flow

The AP is triggered by membrane depolarization that exceeds a certain threshold.The depolarization trigger may result from:

1. Excitatory synaptic input in the dendrites or soma, leading to AP initiation at the base of the axon

2. The action potential at an upstream region of axon, leading to AP propagation along the axon

ACTION POTENTIAL INITIATION:SODIUM CHANNEL DENSITY AT BASE OF AXON AND CHANNEL GATING KINETICS

CREATE A TRIGGER ZONE FOR LARGE INWARD CURRENT

KANDEL FIG 9-6

When excitatory synaptic currents depolarizecell enough to activate small percentage of

sodium channels, high channel density at initial segment gives sufficient

inward sodium current to further depolarizeregion, thereby opening more channels

more rapidly ----> ----->Trigger for explosive opening of all sodiumchannels, large inward currents, and rapid

swing in Vm to positive value.

DEPOLARIZE

DEPOLARIZE

DEPOLARIZE

CHANNELSOPEN

CHANNELSOPEN

SODIUMCURRENT

SODIUMCURRENT

VIEWING ACTION POTENTIAL BY WHOLE-CELL PATCH CLAMPSIZE OF EXCITATORY INPUT (SYNAPTIC) CURRENT DETERMINES SPEED OF INITIATION

GRANULE NEURON IN CEREBELLUM FIRES ACTION POTENTIALS SOONERWITH GREATER INPUT DEPOLARIZING CURRENT

15050 100

CURRENT CLAMP

Current clamp consists of a current generator which commands a specifiedcurrent that runs through the patch pipette back to bath ground,

i.e., across the cell membrane.

Instrument also records voltage from pipette to ground = Vmembrane

+-patchpipet

CELL

bath(grounded)

CURRENTCURRENTSOURCESOURCE

ICOMMAND

VOLTAGEVOLTAGEMONITORMONITOR

Icap

Imem

ground

ICOMMAND

ICOMMAND Imem Icap= +

ICOM 0 pA

+ pA

VMEM Vrest

ACTION POTENTIAL DOWNSTROKESODIUM CHANNEL INACTIVATION AND POTASSIUM CHANNEL ACTIVATION

SODIUM CHANNELS INACTIVATEPOTASSIUM OUTWARD CURRENT

NOTE HYPERPOLARIZATIONCurrents analyzed by V-clamp

KANDEL FIGURE 9-3

HYPERPOLARIZATION OF DOWNSTROKE REQUIRED FOR RECOVERY OF SODIUM CHANNELS AND THEIR AVAILABILITY

FOR RE-FIRING

CONDUCTION ALONG UNMYELINATED AXON: REVISITED

ONE-WAY CONDUCTION OF ACTION POTENTIAL DOWN AXON

SPEED OF CONDUCTION DETERMINED BY RaxialCmembrane TIME CONSTANT OF AXON.THIN AXONS CONDUCT AT ~ 1 mm/msec

WHY DOESN’T ACTION POTENTIAL ATPOINT “C” RETRIGGER A SECOND

ACTION POTENTIAL AT “A”,WHERE CHANNELS HAVE RETURNED TO

RESTING STATE?

BECAUSE POTASSIUM CHANNELS STILL OPEN AT POINT “B” PROVIDE A

SHORT CIRCUIT AGAINST BACK PROPAGATION

A

B

C

D

msec2 4 6 8 1012

CONDUCTION ALONG UNMYELINATED AXON: POTASSIUM CHANNEL SHUNT PREVENTS BACK PROPAGATION

+ + - -- -

- -- -+ ++ ++ +

Point AREST

Point DREST

Point CSODIUM

CURRENT

Point BPOTASSIUM

CURRENT

leak

leak

leak

leak

Na Na Na Na KKKK

RaxialRaxial Raxial

Vm0

-70

A B D EC

Since gaxial > gleak ,

point D undergoes significant passive depolarization

leading to AP

Since gK (at B) >> gaxial ,

point B (and point A) do notundergo much

passive depolarization Back inhibition Forward propagation

BLOCKING POTASSIUM CHANNELS CAUSES BACKFIRING/REFIRING OF ACTION POTENTIALS

OTHER CHANNELS AND CURRENTS MODIFYTHE INTRINSIC FIRING PROPERTIES OF NEURONS

KANDEL FIGURE 9-11

Activation And Inactivation Voltage DependenceActivation And Inactivation Voltage DependenceAnd Kinetics Determine Time Window And Kinetics Determine Time Window

For Channel ConductanceFor Channel Conductance

RATERATE

MEMBRANE VOLTAGE (mV)MEMBRANE VOLTAGE (mV)- 70- 70 - 35- 35 00

ACTIVATION

INACTIVATIONINACTIVATION

FGF-HOMOLOGOUS FACTORS (FHFs):A FAMILY OF NEURONAL PROTEINS THAT BIND SODIUM CHANNELS

Induce long-term,use-dependent

channel inactivation

Raise voltage at whichintrinsic fast inactivation

of channels occursControl

neuronalexcitability

FHFsFHFs Cytoplasmic Subunits Modulating Sodium Channel InactivationCytoplasmic Subunits Modulating Sodium Channel Inactivation

FHF Genes, Isoforms and ExpressionFHF Genes, Isoforms and Expression

-trefoil core ~150 aa-trefoil core ~150 aa 25-30 aa25-30 aaFHF1AFHF1B

FHF2AFHF2B

FHF4AFHF4B

66 aa66 aa

62 aa62 aa

64 aa64 aa

4 aa4 aa

9 aa9 aa

69 aa69 aa

FHFs are broadly expressed in neurons of CNS and PNS.FHFs are broadly expressed in neurons of CNS and PNS.

Generally, different classes of neurons express different profile of FHFsGenerally, different classes of neurons express different profile of FHFs

FHF expression commences during neuronal maturation and isFHF expression commences during neuronal maturation and is stably maintainedstably maintained

Sodium Channels in Sodium Channels in Fhf1Fhf1-/--/-Fhf4Fhf4-/--/- Granule Cells Granule CellsInactivate at More Negative Voltage and Inactivate at More Negative Voltage and

Inactivate Faster At Specific VoltagesInactivate Faster At Specific Voltages

V1/2 = -59.1 +/- 4.8 mV

n = 8 cells V1/2 = -72.8 +/- 4.3 mV

n = 9 cells P < 10P < 10-4-4

(from Goldfarb et atl, Neuron, 2007)

WT FHF1+4 KO

KO

KO

WT

WT

Voltage Dependence Time Constants at Specific Voltages

Fhf1Fhf1-/--/-Fhf4Fhf4-/--/- Granule Cells In Cerebellar Slices CannotGranule Cells In Cerebellar Slices CannotFire Repetitively In Response To Sustained Current InjectionFire Repetitively In Response To Sustained Current Injection

(from Goldfarb et atl, Neuron, 2007)

WHOLE CELL PATCH-CLAMPED GRANULE NEURONSIN ADULT MOUSE CEREBELLUM SLICES

SODIUM CHANNELS INACTIVATE AT MORE NEGATIVE POTENTIALSODIUM CHANNELS INACTIVATE AT MORE NEGATIVE POTENTIAL IN FHF MUTANT NEURONIN FHF MUTANT NEURON

Fhf1Fhf1-/--/-Fhf4Fhf4-/--/-Wild TypeWild Type

WHOLE CELL PATCH-CLAMPED GRANULE NEURONSIN ADULT MOUSE CEREBELLUM SLICES

IMPAIRED SODIUM CHANNEL RECOVERY IN FHF MUTANT NEURONIMPAIRED SODIUM CHANNEL RECOVERY IN FHF MUTANT NEURONWild TypeWild Type Fhf1Fhf1-/--/-Fhf4Fhf4-/--/-

Normal sodium channel density and activation in mutant cells

Current-induced depolarization gives rapid 1st action potentialCurrent-induced depolarization gives rapid 1st action potential

In mutant cells, downstroke of action potential does not lower voltage far enough for many sodium channels to recover from

inactivation, and the rate of channel recovery is impaired

Subsequent action potentials blocked; no repetitive firingSubsequent action potentials blocked; no repetitive firing

ALTERED SODIUM CHANNEL RESPONSES IN Fhf1ALTERED SODIUM CHANNEL RESPONSES IN Fhf1-/--/-Fhf4Fhf4-/--/- GRANULE CELLS CAUSES IMPAIRED EXCITABILITYGRANULE CELLS CAUSES IMPAIRED EXCITABILITY

““A-type” FHFs Induce Long-Term Inactivation of Sodium ChannelsA-type” FHFs Induce Long-Term Inactivation of Sodium Channels

FHF

Isoform

Upshift in V1/2 Steady State Inactivation

Induction of Long-Term Inactivation

1A 13 mV ++++

2A 13 mV ++++++

4A 16 mV ++++++

1B 1 mV -

2B 7 mV -

4B 17 mV -( from Dover et al, J. Physiology, 2010)

Does Channel Fast Inactivation Limit Long-term Inactivation?Does Channel Fast Inactivation Limit Long-term Inactivation?

Mutant Channel DeficientMutant Channel DeficientFor Fast InactivationFor Fast Inactivation

FHF2A Restores InactivationFHF2A Restores InactivationAnd Augments Long-Term InactivationAnd Augments Long-Term Inactivation

( from Dover et al, J. Physiology, 2010)

Long-Term Inactivation Requires FHF2A Channel-BindingLong-Term Inactivation Requires FHF2A Channel-Bindingand N-Terminal Effector Domainsand N-Terminal Effector Domains

( from Dover et al, J. Physiology, 2010)

Long-Term Inactivation Gating Particle ModelLong-Term Inactivation Gating Particle Model

Antibody Inhibition of Channel Long-Term InactivationAntibody Inhibition of Channel Long-Term Inactivation

( from Dover et al, J. Physiology, 2010)

FHF N-Terminal Peptide Injection Recapitulates Long-Term InactivationFHF N-Terminal Peptide Injection Recapitulates Long-Term Inactivation

( from Dover et al, J. Physiology, 2010)