LECTURE 6 ACTION POTENTIAL INITIATION AND PROPAGATION REQUIRED

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 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 3. 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 SODIUM CURRENT DEPOLARIZE CHANNELS OPEN 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 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 = CURRENT SOURCE ICOMMAND patch pipet + Icap m + p. A ground ICO VOLTAGE MONITOR Ime 0 p. A M Ime + ICOMMAND- CELL bath (grounded) m VMEM Icap Vrest

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

CONDUCTION ALONG UNMYELINATED AXON: REVISITED A ONE-WAY CONDUCTION OF ACTION POTENTIAL DOWN AXON SPEED OF CONDUCTION DETERMINED BY Raxial. Cmembrane TIME CONSTANT OF AXON. THIN AXONS CONDUCT AT ~ 1 mm/msec B 2 4 6 8 1012 msec C WHY DOESN’T ACTION POTENTIAL AT POINT “C” RETRIGGER A SECOND ACTION POTENTIAL AT “A”, WHERE CHANNELS HAVE RETURNED TO RESTING STATE? D BECAUSE POTASSIUM CHANNELS STILL OPEN AT POINT “B” PROVIDE A SHORT CIRCUIT AGAINST BACK PROPAGATION

CONDUCTION ALONG UNMYELINATED AXON: POTASSIUM CHANNEL SHUNT PREVENTS BACK PROPAGATION Point B POTASSIUM CURRENT Point A REST Point C SODIUM CURRENT Point D REST ++ ++ -- -- ++ -K a D N C ak Since g. K (at B) >> gaxial , point B (and point A) do not undergo much passive depolarization Vm B Raxial le Since gaxial > gleak , point D undergoes significant passive depolarization leading to AP K A a N ak le Raxial K a N ak le Raxial E 0 -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 0 MEMBRANE VOLTAGE (m. V)

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

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

FHF Genes, Isoforms and Expression 66 aa FHF 1 A FHF 1 B FHF 2 A FHF 2 B FHF 4 A FHF 4 B b-trefoil core ~150 aa 25 -30 aa 4 aa 62 aa 9 aa 64 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 Fhf 1 -/-Fhf 4 -/- 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 FHF 1+4 KO V 1/2 = -59. 1 +/- 4. 8 m. V V 1/2 = -72. 8 +/- 4. 3 m. V n = 8 cells n = 9 cells P < 10 -4 (from Goldfarb et atl, Neuron, 2007)

Fhf 1 -/-Fhf 4 -/- 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 Fhf 1 -/-Fhf 4 -/-

WHOLE CELL PATCH-CLAMPED GRANULE NEURONS IN ADULT MOUSE CEREBELLUM SLICES IMPAIRED SODIUM CHANNEL RECOVERY IN FHF MUTANT NEURON Wild Type Fhf 1 -/-Fhf 4 -/-

ALTERED SODIUM CHANNEL RESPONSES IN Fhf 1 -/-Fhf 4 -/GRANULE CELLS CAUSES IMPAIRED EXCITABILITY Normal sodium channel density and activation in mutant cells Current-induced depolarization gives rapid 1 st 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 ( from Dover et al, J. Physiology, 2010) FHF Isoform Upshift in V 1/2 Steady State Inactivation Induction of Long-Term Inactivation 1 A 13 m. V ++ 2 A 13 m. V +++ 4 A 16 m. V +++ 1 B 1 m. V - 2 B 7 m. V - 4 B 17 m. V -

Does Channel Fast Inactivation Limit Long-term Inactivation? Mutant Channel Deficient For Fast Inactivation FHF 2 A Restores Inactivation And Augments Long-Term Inactivation ( from Dover et al, J. Physiology, 2010)

Long-Term Inactivation Requires FHF 2 A 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)