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Ion Channels and the Regulation of Membrane Potential

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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 KCl KCl Cl- K+ - + membrane potential depends on ionic gradients
--no membrane potential KCl KCl KCl Cl- Cl- --membrane potential K+ K+ - + 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 Cl- K+ - + at equilibrium 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 in the cell membrane equivalent circuit K+ in out + - 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 if multiple channels, which one wins out?
RK RNa RCl EK ENa ECl out in 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 KcsA (bacterial K+ channel)
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+ cation does not interact with charged residues--why?

10 Na+ permeates semi-hydrated
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 depolarization activates both Na+ and K+ channels
(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 Na+ channel
(Bezanilla, 2008) Na+ channel basic residues aligned along one helix movement of voltage sensor can be measured directly

15 gating charge movement
in Na+o = 0 and Na+ channel pore blocker TTX: depolarization gating charge Na+ current 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 distinguish signal from noise?
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 K+ most channels conduct equally in both directions
out in K+ but some conduct more in one direction than the other --rectification

32 K+ inwardly rectifying K+ channels --K+ enters much faster than leaves
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 cytoplasm contains factors that promote rectification
(Vandenberg, 1987) cytoplasm contains factors that promote rectification

34 K+ cannot displace Mg++, only with K+ efflux --rectification
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 µM 6) Vm also drives conformational changes in other membrane proteins --transporters (in what was probably its original role)

38 Channels Molecular Biology of the Cell, Chapter 11. Hille, B. Ion Channels of Excitable Membranes. Sinauer. Aggarwal SK, MacKinnon R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron. 1996;16(6): Bezanilla F. How membrane proteins sense voltage. Nat Rev Mol Cell Biol. 2008;9(4): Bichet D, Haass FA, Jan LY. Merging functional studies with structures of inward-rectifier K(+) channels. Nat Rev Neurosci. 2003;4(12): Doyle DA, Morais Cabral J, Pfuetzner RA, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998;280(5360):69-77. Eggermann, E., Bucurenciu, I., Goswami, S.P., and Jonas, P. (2012). Nanodomain coupling between ca(2)(+) channels and sensors of exocytosis at fast mammalian synapses. Nature reviews. Neuroscience 13, 7-21. Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990;250(4980):533-8. Mayer ML. Glutamate receptors at atomic resolution. Nature. 2006;440(7083): Nishida M, MacKinnon R. Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution. Cell. 2002;111(7): Oliver D, Lien CC, Soom M, Baukrowitz T, Jonas P, Fakler B. Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids. Science. 2004;304(5668): Schwappach B, Zerangue N, Jan YN, Jan LY. Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter. Neuron. 2000;26(1): Seoh SA, Sigg D, Papazian DM, Bezanilla F. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron. 1996;16(6): Sobolevsky AI, Rosconi MP, Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462: Sudhof, T.C. (2012). The presynaptic active zone. Neuron 75, Sun Y, Olson R, Horning M, Armstrong N, Mayer M, Gouaux E. Mechanism of glutamate receptor desensitization. Nature. 2002;417(6886):

39 Tao X, Avalos JL, Chen J, MacKinnon R (2009) Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution. Science 326: Vandenberg CA. Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions. Proc Natl Acad Sci U S A. 1987;84(8): Wilson CJ. The mechanism of intrinsic amplification of hyperpolarizations and spontaneous bursting in striatal cholinergic interneurons. Neuron. 2005;45(4): Yang N, Horn R. Evidence for voltage-dependent S4 movement in sodium channels. Neuron. 1995;15(1):213-8. Zagotta WN, Hoshi T, Aldrich RW. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science. 1990;250(4980):

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