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

2. 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

3. 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

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

5. Voltage sensing Transmembrane potential stabilizes S4 S4 moves S5/S6 Pore open/close 2 1 3 4 5 6

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

7. 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

8. 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

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

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

11. 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

12. 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”

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

14. 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

15. 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+

16. Na V causes local depolarization Membrane capacitance of 10 -6 F/cm 2 –10 -6 (  r 2 ) Na influx: n (1.6 10 -19 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

17. 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

18. 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

19. 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

20. 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”

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

22. N-type calcium channels Neurotransmitter release (presynaptic) –Calcium dynamics same time scale as firing (10 ms) –Highly localized changes (50-5000 nm) Post-synaptic, Ca-dependent remodeling

23. Striated Muscle Cardiac Skeletal “Twitch” force –50-200 ms –All-or-none Tension depends on [Ca 2+ ] I –Spontaneous –Neural

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

25. 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

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

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