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What is membrane potential? membrane potential is the charge difference across a membrane at the plasma membrane, Vm = Vin - Vout --all membranes express.

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Presentation on theme: "What is membrane potential? membrane potential is the charge difference across a membrane at the plasma membrane, Vm = Vin - Vout --all membranes express."— Presentation transcript:

1 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 Ion Channels and the Regulation of Membrane Potential

2 KCl --no membrane potential KCl K+K+ K+K+ Cl 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 membrane potential depends on ionic gradients --membrane potential

3  G conc +  G volt = 0  G conc = -RT ln C o /C i  G volt = zFV where z= valence of ion zFV – RT ln C o /C i = 0 V = RT ln C o /C i = 2.3 RT log 10 C o /C i zF zF since 2.3 RT/F ~60 mV, V = (60 mV/z) log 10 (C o /C i ) no net flux of K + : influx = efflux membrane potential is equal and opposite to the concentration gradient at equilibrium KCl K+K+ K+K+ Cl - - +

4 in out in K+K+ K+K+ + - membraneequivalent circuit in the cell

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 RKRK R Na R Cl EKEK E Na E Cl out in if multiple channels, which one wins out? --the biggest one (or the most open channels with that selectivity), driving Vm to the equilibrium potential for that ion Goldman-Hodgkin-Katz eqn

7 channels selective for different ions control Vm K + channels drive cell to E K Na + channels drive cell to E Na equal numbers of K + and Na + channels drive cell to potential between E K and E Na (GHK eqn) --calculate Erev (reversal potential) for p K 0.5, p Na 0.5 channels equally selective for K + and Na + also drive cell to potential between E Na and E K 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 (Doyle et al, 1998) KcsA (bacterial K + channel) tetramer with 2 TMD/subunit inverted teepee

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

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

11 gating membrane potential ion channels permeation gating

12 (TC Sudhof, 2008) action potential

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

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

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

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

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

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

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 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 (Sun et al, 2002)

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 out in but some conduct more in one direction than the other --rectification K+K+ K+K+

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

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

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

35 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 several kinds of inward rectifiers IRKs K ATP G protein-coupled (GIRKs)

36 Conclusions membrane potential ion channels permeation gating 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): 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, Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990;250(4980): 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): 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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