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

membraneat 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 metabolitesthe flux of inorganic ions

Ion Channels and the Regulation of Membrane Potential

KClKCl --no membrane potential

KClKCl

K+K+

Cl-Cl-

-+ http://nerve.bsd.uchicago.edu/

key features: very few ions needed to generate membrane potential (fast, cheap)

does not change bulk concentrationssystem comes to rest at a new equilibrium

membrane potential depends on ionic gradients

--membrane potential

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)

no net flux of K+: influx = efflux

membrane potential is equal and opposite to the concentration gradient

at equilibrium

KClKCl

K+K+

Cl-Cl-

-+

in

out

inK+

K+

+

-

membrane equivalent circuit

in the cell

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 eqn3) how does the valence of the ion affect equilibrium potential?

intuitively, why would this be (use Ca++ as example)?

RK RNa RCl

EK ENa ECl

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

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)

membrane potential

ion channels permeation gating

selectivity

(Doyle et al, 1998)

KcsA (bacterial K+ channel)

tetramer with 2 TMD/subunitinverted teepee

hydration of ion replaced by backbone carbonyls --less effective for smaller Na+

cation does not interact with charged residues--why?

recognition of dehydrated ion

Na+ channel

smaller ionbigger poreNa+ permeates semi-hydrated

(Payandeh et al., 2011)

gating

membrane potential

ion channels permeation gating

(TC Sudhof, 2008)

acti

on

po

ten

tial

depolarization activates both Na+ and K+ channels but with slight lag between opening of eachfirst Na+ (driving Vm to ENa), then K+ channels (driving Vm to EK) --positive then negative feedbackthen channels inactivatemediates lateral propagation of depolarization across membrane

(Bezanilla, 2008)

voltage sensor

K+ channel

Na+ channel

(Bezanilla, 2008)

basic residues aligned along one helixmovement of voltage sensor can be measured directly

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

neutralization of voltage sensor reduces gating charge12-16 charges/channel (3-4/subunit)

(Aggarwal and MacKinnon, 1996)(Seoh et al, 1996)

biochemical evidence: replace charged residues in voltage sensor

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)

inactivation

inactivation not required to restore resting Vm --what is inactivation good for?

channels inactivate with a characteristic delaywhat controls inactivation?

proteolysis reduces fast inactivation (N-type)

(Hoshi et al, 1990; Zagotta et al, 1990)

inactivation is not the opposite of activationreactivation requires two transitionsit is a distinct process triggered by conformational changes --characteristic delay encoded by protein

(TC Sudhof, 2008)

steep dependence on [Ca++]o (synaptotagmin)nano-domain coupling:

(Eggermann et al., 2011)

mechanism

wt

RIM DKO

tighter coupling = enhanced synchrony

(TC Sudhof, 2008)

dimer of dimers

upright teepee

glutamate binds at D1-D2 interface, within monomer

(Sobolevsky et al., 2009)

AMPA receptor

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?

inactivation is one method to terminate signaling

channel closes rapidly in continued presence of glutamatedepends on weakening of D1-D1 interface (wt Kd ~6 mM)L483Y mutant Kd 0.03 µM--little desensitization

(Sun et al, 2002)

desensitization

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

how can a neurondistinguish signal from noise?

--short EPSPs(excitatory postsynaptic potentials)require summation to reachthreshold, trigger action potential

10 pA2 s

most channels conduct equally in both directions

out

in

but some conduct more in one directionthan the other--rectification

K+

K+

inwardly rectifying K+ channels

--K+ enters much faster than leaves

returns Vm to EK for small depolarizationinactivates for large depolarizations--how?

out

in

(Bichet et al, 2003)

K+

K+

cytoplasm contains factors that promote rectification

(Vandenberg, 1987)

(Tao et al., 2009)

Rb+ (~K+) Sr++ (~Mg++)

K+ cannot displace Mg++, only with K+ efflux--rectification

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+ channelshyperpolarization-activated channels (Na+, Ca++)

--contribute to oscillatory behavior

several kinds of inward rectifiersIRKsKATP

G protein-coupled (GIRKs)

Conclusions

membrane potentialion channels permeation gating

1) Membrane potential (Vm) is determined by ionic gradients and the relative permeability of different ions2) Very few ions need to flow to change membrane potential 3) Channels drive Vm to the equilibrium potential of their permeant ions4) Permeability is determined by relatively weak interactions and mutual repulsion of ions in the pore5) 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 timing7) Channels can rectify, improving signal/noise8) Channels can be used to process information (switch-like cooperative responses, coincidence detection, oscillators) within single cells

what do the changes in Vm accomplish?

1) propagate signal down axon

2) trigger transmitter release at terminal3) activate contractile apparatus in muscle4) 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)

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):1169-77.Bezanilla F. How membrane proteins sense voltage. Nat Rev Mol Cell Biol. 2008;9(4):323-32.Bichet D, Haass FA, Jan LY. Merging functional studies with structures of inward-rectifier K(+) channels. Nat Rev Neurosci. 2003;4(12):957-67.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):456-62.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):957-65.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):265-70.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):155-67.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):1159-67.Sobolevsky AI, Rosconi MP, Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462:745-756.Sudhof, T.C. (2012). The presynaptic active zone. Neuron 75, 11-25.Sun Y, Olson R, Horning M, Armstrong N, Mayer M, Gouaux E. Mechanism of glutamate receptor desensitization. Nature. 2002;417(6886):245-53.

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 326:1668-1674.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):2560-4.Wilson CJ. The mechanism of intrinsic amplification of hyperpolarizations and spontaneous bursting in striatal cholinergic interneurons. Neuron. 2005;45(4):575-85.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):568-71.