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 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 DEPOLARIZE CHANNELS OPEN SODIUM CURRENT When excitatory synaptic currents depolarize cell enough to activate small percentage of sodium channels, high channel density at initial segment gives sufficient inward sodium current to further depolarize region, thereby opening more channels more rapidly ----> -----> Trigger for explosive opening of all sodium channels, large inward currents, and rapid swing in Vm to positive value.

VIEWING ACTION POTENTIAL BY WHOLE-CELL PATCH CLAMP SIZE OF EXCITATORY INPUT (SYNAPTIC) CURRENT DETERMINES SPEED OF INITIATION 50 100 150 GRANULE NEURON IN CEREBELLUM FIRES ACTION POTENTIALS SOONER WITH GREATER INPUT DEPOLARIZING CURRENT

- CURRENT CLAMP ICOMMAND Imem Icap = + ICOMMAND ICOM Imem + VMEM Vrest Current clamp consists of a current generator which commands a specified current 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 ICOMMAND Imem Icap = + CURRENT SOURCE ICOMMAND + pA ground ICOM 0 pA VOLTAGE MONITOR bath (grounded) Imem + - patch pipet VMEM Vrest ICOMMAND Icap CELL

ACTION POTENTIAL DOWNSTROKE SODIUM CHANNEL INACTIVATION AND POTASSIUM CHANNEL ACTIVATION KANDEL FIGURE 9-3 SODIUM CHANNELS INACTIVATE POTASSIUM OUTWARD CURRENT NOTE HYPERPOLARIZATION Currents analyzed by V-clamp HYPERPOLARIZATION OF DOWNSTROKE REQUIRED FOR RECOVERY OF SODIUM CHANNELS AND THEIR AVAILABILITY FOR RE-FIRING

A B C D 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 AT POINT “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 2 4 6 8 10 12 msec

CONDUCTION ALONG UNMYELINATED AXON: POTASSIUM CHANNEL SHUNT PREVENTS BACK PROPAGATION Point B POTASSIUM CURRENT Point C SODIUM CURRENT Point A REST Point D REST - - + + + + + + - - - - - - + + leak Na K Raxial leak Na K Raxial leak Na K Raxial leak Na K A B C D E Since gaxial > gleak , point D undergoes significant passive depolarization leading to AP Since gK (at B) >> gaxial , point B (and point A) do not undergo much Vm -70 Back inhibition Forward propagation BLOCKING POTASSIUM CHANNELS CAUSES BACKFIRING/REFIRING OF ACTION POTENTIALS

OTHER CHANNELS AND CURRENTS MODIFY THE INTRINSIC FIRING PROPERTIES OF NEURONS KANDEL FIGURE 9-11

Activation And Inactivation Voltage Dependence And Kinetics Determine Time Window For Channel Conductance ACTIVATION RATE INACTIVATION - 70 - 35 MEMBRANE VOLTAGE (mV)

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

Cytoplasmic Subunits Modulating Sodium Channel Inactivation FHFs Cytoplasmic Subunits Modulating Sodium Channel Inactivation Induce long-term, use-dependent channel inactivation Raise voltage at which intrinsic fast inactivation of channels occurs Control neuronal excitability

FHF Genes, Isoforms and Expression b-trefoil core ~150 aa 25-30 aa FHF1A FHF1B FHF2A FHF2B FHF4A FHF4B 66 aa 62 aa 64 aa 4 aa 9 aa 69 aa FHFs are broadly expressed in neurons of CNS and PNS. Generally, different classes of neurons express different profile of FHFs FHF expression commences during neuronal maturation and is stably maintained

Sodium Channels in Fhf1-/-Fhf4-/- Granule Cells Inactivate at More Negative Voltage and Inactivate Faster At Specific Voltages Voltage Dependence Time Constants at Specific Voltages KO WT WT KO WT FHF1+4 KO V1/2 = -59.1 +/- 4.8 mV n = 8 cells V1/2 = -72.8 +/- 4.3 mV n = 9 cells P < 10-4 (from Goldfarb et atl, Neuron, 2007)

Fhf1-/-Fhf4-/- Granule Cells In Cerebellar Slices Cannot Fire Repetitively In Response To Sustained Current Injection (from Goldfarb et atl, Neuron, 2007)

WHOLE CELL PATCH-CLAMPED GRANULE NEURONS IN ADULT MOUSE CEREBELLUM SLICES SODIUM CHANNELS INACTIVATE AT MORE NEGATIVE POTENTIAL IN FHF MUTANT NEURON Wild Type Fhf1-/-Fhf4-/-

WHOLE CELL PATCH-CLAMPED GRANULE NEURONS IN ADULT MOUSE CEREBELLUM SLICES IMPAIRED SODIUM CHANNEL RECOVERY IN FHF MUTANT NEURON Wild Type Fhf1-/-Fhf4-/-

ALTERED SODIUM CHANNEL RESPONSES IN Fhf1-/-Fhf4-/- GRANULE CELLS CAUSES IMPAIRED EXCITABILITY Normal sodium channel density and activation in mutant cells Current-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 firing

“A-type” FHFs Induce Long-Term Inactivation of Sodium Channels Isoform Upshift in V1/2 Steady State Inactivation Induction of Long-Term Inactivation 1A 13 mV ++ 2A +++ 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? Mutant Channel Deficient For Fast Inactivation FHF2A Restores Inactivation And Augments Long-Term Inactivation ( from Dover et al, J. Physiology, 2010)

Long-Term Inactivation Requires FHF2A Channel-Binding and N-Terminal Effector Domains ( from Dover et al, J. Physiology, 2010)

Long-Term Inactivation Gating Particle Model

Antibody Inhibition of Channel Long-Term Inactivation ( from Dover et al, J. Physiology, 2010)

FHF N-Terminal Peptide Injection Recapitulates Long-Term Inactivation ( from Dover et al, J. Physiology, 2010)