From Hodgkin (1957)

Ca2+-dependent action potentials

Ca2+ action potential properties (Hagiwara & Byerly, 1978) Overshoot or rate of rise varies with Cao (29 mV per 10-fold) AP disappears when Mg replaces Ca Overshoot and rate of rise don’t change when Nao removed AP persists when Ca replaced with Sr or Ba AP blocked by metal cations: Co2+, La3+, Mn2+, Cd2+, Ni2+

Ca2+ currents are more difficult to study than Na+ currents Current separation not simple: Ca2+ current overlaps with K+ current K+ current often depends on ICa (Ca2+-activated K+ current) No defined Ca2+ equilibrium potential: ECa not measurable can’t use I = g(V-ECa) Inactivation is complex: depends on current as well as voltage Voltage-dependent facilitation: repetitive voltage steps can increase current Dependence on phosphorylation: washout during internal perfusion (NT changes with time)

Na+ current Ca2+ current [Ca2+]o/[Ca2+]i = ~10,000 [Na+]o/[Na+]i = ~15 Erev = ~+50 mV Erev not measurable conductance is f(V, Ca2+) linear conductance Ion saturation and block Ion fluxes are independent

Ca2+ conductance is not linear Rectification: Ca2+ equilibrium potential not measurable conductance large at negative Vm conductance small at positive Vm [Ca2+]inexp(2VF/RT)-[Ca2+]o iCa(V) = PCa 4F2 V RT exp(2VF/RT) - 1

How to get high selectivity and high flux rate Problem: How to get high selectivity and high flux rate Na+ Na+ ionic radius 1.14 Å Ca2+ Ca2+ ionic radius 1.16 Å [Na]o >> [Ca2+]o iCa = 106 ions/second out in

Rate of rise of Ca action potential Ion saturation indicates Ca2+ binds to the channel Ba2+ Rate of rise of Ca action potential Ca2+ KBa ~ 20 mM KCa ~ 5 mM Concentration (mM) Saturation ICa ~ 1 1 + [Ca2+] KD Ca binds to the channel with mM KD How to reconcile low affinity with high selectivity

Ca2+ channels let Na+ ions through in the absence of Ca2+ (Almers et al, J. Physiol. 353:565, 1984) I-monovalent Cao = 60 nM 30 µM Cao Cao = 30 µM Imonovalent blocked by µM Cao 60 nM Cao High-affinity Ca2+ binding site in channel

Outward current through Ca2+channels carried by monovalent cations (Lee & Tsien, J Physiol. 354:253, 1984) Experiment Out: Ba In: Cs Whole-cell large Iout Cs+ Ba2+

Problem: Low affinity site (millimolar) from saturation of ICa High affinity site (micromolar) from block of monovalent INa+ by Ca2+o

Two binding site model for Ca2+ channel permeation -log[Ca]

Ca2+ currents produce voltage-dependent Ca2+ accumulation snail neuron snail neuron snail neuron salamander photoreceptor squid synapse

Inactivation depends on Ca2+ current

Two-pulse voltage clamp analysis of current-dependent inactivation Ca2+ current during prepulse (normalized to max) Ca2+ current during second test pulse (normalized to max) Ca2+ current during test pulse reduced maximally when prepulse current evokes max Ica (Ba2+ current not affected)

Ion channel diversity: why? Resting potential Complex electrical activity: Pacemaker potentials Burst generation Spike adaptation Resonance Amplification of synaptic responses Coupling electrical activity to neuronal function: Ca2+ entry: neurotransmitter release, growth cone motility, gene expression Calcium waves and oscillations Development Impedance matching

Molecular diversity of voltage-dependent ion channels Name Genes Physiologically defined currents K+ Kv 27 Neuronal delayed rectifier; A-current Eag 8 Cardiac fast ‘delayed rectifier’ KCNQ 5 M-current; cardiac slow ‘delayed rectifier’ SK 4 Low conductance Ca2+-dependent Slo 3 High conductance Ca2+- and voltage-dependent Kir 16 Inward rectifiers, G protein-activated 2P 15 Leakage (resting potential) Na+ TTX-s 6 Neurons, adult skeletal muscle, glia TTX-r Cardiac muscle, some sensory neurons Ca2+ S, C, D, F High voltage-activated (L-type) A, B, E High voltage-activated (N, P/Q, R-types) G, H, I Low voltage-activated (T-type) Cation Pacemaker Cardiac muscle, some neurons CNG Cyclic nucleotide-gated Trp 17 Capacitative Ca2+ entry

Na+ channel diversity TTX-resist. INa fast: H-H kinetics INa persistent: incomplete inactivation INa resurgent: passes through open state during recovery from inactivation (NaV1.6)

Na+ currents in DRG neurons Large afferent Fast TTX-sens. Nav1.6 Small sensory Slow TTX-resist. NaV1.7,8,9 Incomplete inactivation: Vm = -30 m = ~.3 h = ~.3 Small sensory Both contributes to hyperexcitability

Persistent Na+ currents amplify sub-threshold synaptic responses cortical & hippocampal pyramidal cells Persistent INa amplifies synaptic potential hyperpolarize cell

Resurgent Na+ currents in cerebellar Purkinje neurons (Raman & Bean, 1997) fast INa

Resurgent Na+ currents: burst generation R  O  I Resurgent I  O recovery

Ca2+ channel diversity Nifedipine, diltiazem, verapamil Dihydropyridine- sensitive Nifedipine, diltiazem, verapamil Dihydropyridine- insensitive agatoxin IVA, conotoxin MVIIC w-conotoxin GVIA ‘resistant’ P/Q, N, and R: presynaptic localization, transmitter release L-type: postsynaptic localization, cell bodies and dendrites gene expression, cerebellar LTD, plateau potentials

High- and low threshold Ca2+ currents Weak depol Strong depol Large, sustained Inward Ba current LVA - T-type HVA - L, N, P, R

Burst firing in thalamocortical relay neurons: T-type Ca2+ channels (CaV3.1)

Conus striatus - a fish eater

Pharmacological components of neuronal high-threshold Ca2+ currents Acutely isolated cortical & striatal neurons (Mermelstein et al, J. Neurosci. 19:7268, 1999) L P N Q

K+ channel diversity charybdotoxin apamin dendrotoxin tetraethylammonium ion Ba2+

Different K+ currents contribute to action potential complexity

Rapidly inactivating K+ current (IA) (Connors & Stevens, 1971)

IA controls bursting in snail neurons (Connors & Stevens, 1971) IA contributes to maintained hyperpolarization Repolarization removes IA inactivation

Ca2+-activated K+ current (IK-Ca) (Meech, 1970s) mV IK - Cao mV +150 Slope = 29 mV/10-fold ∆Cao IK-+Ca- IK-Ca Er Cao 5 Ca 10 Ca 20 Ca

Ca2+-activated K+ currents contribute to the after-hyperpolarization

Ca2+accumulation and Ca2+-activated K+ current

Resonance in hair cells (Art & Fettiplace, J Physiol 385:207-242, 1987)

Inwardly rectifying K+ currents Hyperpolarization-activated Gating depends on V-EK Rectification due to block by internal Mg2+ Functions: Sets resting potential Augments depolarization & repolarization Protects cells against damage-induced depolarization

Pacemaker channels HCN hyperpolarization-activated Cation-permeable