what is membrane potential? membrane potential is the charge difference across a membrane

1. Ion Channels and the Regulation of Membrane Potential what is membrane potential? membrane potential is the charge difference across a membrane at the plasma membrane, Vm = Vin - Vout --all membranes express membrane potential what produces membrane potential? fixed negative charge inside the cell (DNA, RNA) impermeant cellular metabolites the flux of inorganic ions

2. membrane potential depends on ionic gradients KCl --no membrane potential KCl KCl KCl Cl- Cl- --membrane potential K+ K+ - + http://nerve.bsd.uchicago.edu/ key features: very few ions needed to generate membrane potential (fast, cheap) does not change bulk concentrations system comes to rest at a new equilibrium

3. KCl KCl Cl- at equilibrium Cl- K+ K+ - + no net flux of K+: influx = efflux membrane potential is equal and opposite to the concentration gradient Gconc + Gvolt = 0 Gconc = -RT ln Co/Ci Gvolt = zFV where z= valence of ion zFV – RT ln Co/Ci = 0 V = RT ln Co/Ci = 2.3 RT log10 Co/Ci zF zF since 2.3 RT/F ~60 mV, V = (60 mV/z) log10 (Co/Ci)

4. out in in the cell membrane equivalent circuit K+ + - K+ in

5. 1) use your intuitive understanding to determine the sign of the equilibrium potentials for Na+, K+, Cl- and Ca++ 2) calculate the equilibrium potentials from the Nernst eqn 3) how does the valence of the ion affect equilibrium potential? intuitively, why would this be (use Ca++ as example)?

6. out RK RNa RCl EK ENa ECl in if multiple channels, which one wins out? Goldman-Hodgkin-Katz eqn --the biggest one (or the most open channels with that selectivity), driving Vm to the equilibrium potential for that ion

7. channels selective for different ions control Vm K+ channels drive cell to EK Na+ channels drive cell to ENa equal numbers of K+ and Na+ channels drive cell to potential between EK and ENa (GHK eqn) --calculate Erev (reversal potential) for pK 0.5, pNa 0.5 channels equally selective for K+ and Na+also drive cell to potential between ENa and EK ionic selectivity determines Vm how can K+ channels distinguish between cations? (Na+ is smaller than K+) --defects in selectivity devastating (weaver mutation)

8. membrane potential ion channels permeation gating selectivity KcsA (bacterial K+ channel) (Doyle et al, 1998) tetramer with 2 TMD/subunit inverted teepee

9. recognition of dehydrated ion hydration of ion replaced by backbone carbonyls --less effective for smaller Na+ cationdoes not interact with charged residues--why?

10. Na+ channel (Payandeh et al., 2011) smaller ion bigger pore Na+ permeates semi-hydrated

11. membrane potential ion channels permeation gating gating

12. action potential (TC Sudhof, 2008)

13. (Bezanilla, 2008) depolarization activates both Na+ and K+ channels but with slight lag between opening of each first Na+ (driving Vm to ENa), then K+ channels (driving Vm to EK) --positive then negative feedback then channels inactivate mediates lateral propagation of depolarization across membrane

14. voltage sensor K+ channel (Bezanilla, 2008) Na+ channel basic residues aligned along one helix movement of voltage sensor can be measured directly

15. depolarization gating charge Na+ current gating charge movement in Na+o = 0 and Na+ channel pore blocker TTX: note size and temporal relationship between currents

16. biochemical evidence: replace charged residues in voltage sensor (Aggarwal and MacKinnon, 1996) (Seoh et al, 1996) neutralization of voltage sensor reduces gating charge 12-16 charges/channel (3-4/subunit)

17. how does voltage sensor move? if membrane potential concentrated across shallow part of the protein, does not need to move far (Bezanilla, 2008) S4 (blue) rotates, pulling S6 (magenta) to open pore

18. inactivation inactivation not required to restore resting Vm --what is inactivation good for? channels inactivate with a characteristic delay what controls inactivation?

19. proteolysis reduces fast inactivation (N-type) (Hoshi et al, 1990; Zagotta et al, 1990)

20. inactivation is not the opposite of activation reactivation requires two transitions it is a distinct process triggered by conformational changes --characteristic delay encoded by protein

21. (TC Sudhof, 2008)

22. steep dependence on [Ca++]o (synaptotagmin) nano-domain coupling: (Eggermann et al., 2011)

23. mechanism wt RIM DKO

24. tighter coupling = enhanced synchrony

25. (TC Sudhof, 2008)

26. AMPA receptor dimer of dimers upright teepee glutamate binds at D1-D2 interface, within monomer (Sobolevsky et al., 2009)

27. how does glutamate binding open the channel? crystal structures of soluble domain suggest domain closure by ligand --pulls pore-lining helices apart how can receptors respond to high-frequency release?

28. inactivation is one method to terminate signaling (Sun et al, 2002) channel closes rapidly in continued presence of glutamate depends on weakening of D1-D1 interface (wt Kd ~6 mM) L483Y mutant Kd 0.03 µM--little desensitization

29. desensitization weakened D1-D1 interface relieves strain on D2, allowing channel closure

30. how can a neuron distinguish signal from noise? --short EPSPs (excitatory postsynaptic potentials) require summation to reach threshold, trigger action potential 10 pA 2 s

31. most channels conduct equally in both directions K+ out in K+ but some conduct more in one direction than the other --rectification

32. K+ out inwardly rectifying K+ channels --K+ enters much faster than leaves K+ in (Bichet et al, 2003) returns Vm to EK for small depolarization inactivates for large depolarizations--how?

33. (Vandenberg, 1987) cytoplasm contains factors that promote rectification

34. Rb+ (~K+) Sr++ (~Mg++) (Tao et al., 2009) K+ cannot displace Mg++, only with K+ efflux --rectification

35. several kinds of inward rectifiers IRKs KATP G protein-coupled (GIRKs) after depolarization inactivates IRK, all-or-none response positive feedback: voltage-activated Na+ or Ca++ channels amplify the response, making it switch-like negative feedback: voltage-gated K+ channels hyperpolarization-activated channels (Na+, Ca++) --contribute to oscillatory behavior

36. membrane potential ion channels permeation gating Conclusions 1) Membrane potential (Vm) is determined by ionic gradients and the relative permeability of different ions 2) Very few ions need to flow to change membrane potential 3) Channels drive Vm to the equilibrium potential of their permeant ions 4) Permeability is determined by relatively weak interactions and mutual repulsion of ions in the pore 5) Channels can be gated by extracellular and intracellular ligands and by membrane potential itself 6) Inactivation (including desensitization) involves mechanisms distinct from activation--for precise timing 7) Channels can rectify, improving signal/noise 8) Channels can be used to process information (switch-like cooperative responses, coincidence detection, oscillators) within single cells

37. what do the changes in Vm accomplish? 1) propagate signal down axon 2) trigger transmitter release at terminal 3) activate contractile apparatus in muscle 4) activate signaling proteins (e.g., kinases) 5) regulate gene expression but how can changes in Vm do this if bulk concentrations of Na+, K+, Cl- do not change? --Ca++ very low inside cell (100 nM) voltage-gated Ca++ channels mediate influx from external solution (1 mM) increasing Ca++I to 1-100 µM 6) Vm also drives conformational changes in other membrane proteins --transporters (in what was probably its original role)

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