Voltage gated channels Molecular structure –Na +, K +, Ca ++ –Cl - Voltage sensing Action potential Calcium signaling

Core voltage-gating functional unit 6 transmembrane segments –One charged –Pore facing –Ion selectivity & V- dependence Tetrameric organization –4x separate, 6 pass proteins –1 protein with 4, 6 pass domains Transmembrane domain PDB: 2r9r Potassium channel has 4 separate subunits

Voltage gated sodium channel Ion selectivity and voltage sensitivity from S4 helices Long cytoplasmic loops btw domains Intracellular domains subject to modification –Conductivity –Open probability Sodium channel has 4 functional domains

Domain organization Common prokaryotic ancestor –S5-S6 4 subunit/domain Pore forming motif Organization S1 S2 S3 S4 S5-6 Canonical subunit K+ structure

Voltage sensing Transmembrane potential stabilizes S4 S4 moves S5/S6 Pore open/close

Chloride Channel Double barreled, 2 subunit channel Each subunit has 3 charged helices with anti-parallel arrangement forming V-sensor PDB: 1kpl

Whole cell recording Clamp voltage Record current Aggregate channel activity & density G=1/R=I/V Applied V Time Current Recorded I Derived I-V Derived Conductance Rectification: Current diverges from straight-line conductance CO V State Model

Channel Inactivation Feedback mechanism Membrane depolarization –Reduces driving force –Secondary conformational change Depolarization Voltage steps Preconditioned Depolarized Channel opens with depolarization Channel becomes refractory with depolarization CO V I State Model

State transitions with voltage clamp C O V I C O V I C O V I C O V I

Characteristics of voltage gated channel Conductance Ion selectivity Threshold Open time Inactivation time

Anatomy of Action potential Voltage gated channels selectively drive intracellular potential between different ionic equilibrium potentials –K+ -90mV –Na+ +60mV Threshold for V-gated Na+ channels Neural AP Cardiac AP

Ionic currents in AP Step voltage to increasing depolarization Net current Na+ current K+ current Sub-threshold Depolarizing current Inactivates Large depolarization opens new K+ channels “Delayed rectifier”

Ionic currents in AP Current declines over time, even though potential remains constant

Ionic contributions to AP K leak (Kir) set resting potential –Inactivate at threshold Na V –Open at threshold –Rapid, large g K V –Open at threshold –Delayed rectifier (slow) –Large g

Anatomy of Cardiac AP Leaky membranes (7) give slow depolarization to Threshold opens Ca V (3) & Na V (1) K V (4) and K Ca repolarize Prolonged AP vs neuron –Ca current –Much delayed K+

Na V causes local depolarization Membrane capacitance of F/cm 2 –10 -6 (  r 2 ) Na influx: n ( C) Threshold ~-40mV V=Q/C –20-30 channels/micron 2 –~ 400 ions/channel to depolarize neighbors Na + r -90 mV -40 mV 10 4 ions/  m 2

Equivalent Circuit Borrowed from cable theory –Break cell into parallel compartments –Propagation depends on resistance/capacitance CmCm CmCm Extracellular CmCm CmCm RmRm RmRm RiRi Intracellular

Neural cable theory Neuron size vs conduction velocity –Large diameter, low internal resistance Myelinated/Unmyelinated –Insulates membrane –Increases R M –Decreases C M –Increase V Node of Ranvier

Na V Modulation 10 genes Alternative splicing Phosphorylation Protein binding Alters –Threshold –Conductivity –Kinetics –Selectivity Cn RPTP PKA PKC PKA PKC Cn RPTP Phosphatases increase conduction Kinases decrease conduction -28 identified binding partners Cytoskeletal Adhesion Signaling

Calcium channel Most common effector of AP Same basic structure as other VG channels Major classes –N-type “Neuronal” –L-Type “Long” –T-Type “Tiny”

Neurons Ionotropic = channels Metabotropic = receptors Neurotransmitter release depends on [Ca 2+ ] I –Multiple inputs Nerve terminals & presynaptic vessicles

N-type calcium channels Neurotransmitter release (presynaptic) –Calcium dynamics same time scale as firing (10 ms) –Highly localized changes ( nm) Post-synaptic, Ca-dependent remodeling

Striated Muscle Cardiac Skeletal “Twitch” force – ms –All-or-none Tension depends on [Ca 2+ ] I –Spontaneous –Neural

L-type calcium channels Excitation contraction coupling Long open time (100 ms) Modulation –Calcium dependent inhibition –Oxidation –Phosphorylation

T-Type calcium channels Tiny conductance (6 vs 25 pS) Low threshold (-50 vs -30 mV) Regulatory role –Cell differentiation –Modulation of phenotype –Neuronal bursting

Smooth muscle Tonic –Vascular –Respiratory Phasic –GI –Bladder Tension depends on [Ca 2+ ] I –Hormonal –Mechanical –Neural Smooth muscle cells in vasculature, gut, sphincter

Smooth Muscle Calcium Ligand gated Ca channels Voltage gated Ca channels Second messengers