Presentation on theme: "Ion Channels and the Regulation of Membrane Potential"— Presentation transcript:
1Ion Channels and the Regulation of Membrane Potential what is membrane potential?membrane potential is the charge difference across a membraneat the plasma membrane, Vm = Vin - Vout--all membranes express membrane potentialwhat produces membrane potential?fixed negative charge inside the cell (DNA, RNA)impermeant cellular metabolitesthe flux of inorganic ions
2KCl KCl Cl- K+ - + membrane potential depends on ionic gradients --no membrane potentialKClKClKClCl-Cl---membrane potentialK+K+-+key features:very few ions needed to generate membrane potential (fast, cheap)does not change bulk concentrationssystem comes to rest at a new equilibrium
3KCl Cl- K+ - + at equilibrium no net flux of K+: influx = efflux membrane potential is equal and opposite to the concentration gradientGconc + Gvolt = 0Gconc = -RT ln Co/CiGvolt = zFV where z= valence of ionzFV – RT ln Co/Ci = 0V = RT ln Co/Ci = 2.3 RT log10 Co/CizF zFsince 2.3 RT/F ~60 mV, V = (60 mV/z) log10 (Co/Ci)
4in the cellmembraneequivalent circuitK+inout+-K+in
51) 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)?
6if multiple channels, which one wins out? RKRNaRClEKENaECloutinGoldman-Hodgkin-Katz eqn--the biggest one (or the most open channels with that selectivity),driving Vm to the equilibrium potential for that ion
7channels selective for different ions control Vm K+ channels drive cell to EKNa+ channels drive cell to ENaequal numbers of K+ and Na+ channels drivecell to potential between EK and ENa (GHK eqn)--calculate Erev (reversal potential) for pK 0.5, pNa 0.5channels equally selective for K+ and Na+ alsodrive cell to potential between ENa and EKionic selectivity determines Vmhow can K+ channels distinguish between cations?(Na+ is smaller than K+)--defects in selectivity devastating (weaver mutation)
8KcsA (bacterial K+ channel) membrane potentialion channelspermeationgatingselectivityKcsA (bacterial K+ channel)(Doyle et al, 1998)tetramer with 2 TMD/subunitinverted teepee
9recognition of dehydrated ion hydration of ion replaced by backbone carbonyls--less effective for smaller Na+cation does not interact with charged residues--why?
13depolarization activates both Na+ and K+ channels (Bezanilla, 2008)depolarization activates both Na+ and K+ channelsbut 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
14voltage sensor K+ channel Na+ channel (Bezanilla, 2008)Na+ channelbasic residues aligned along one helixmovement of voltage sensor can be measured directly
15gating charge movement in Na+o = 0 and Na+ channel pore blocker TTX:depolarizationgating chargeNa+ currentnote size and temporal relationship between currents
16biochemical evidence: replace charged residues in voltage sensor (Aggarwal and MacKinnon, 1996)(Seoh et al, 1996)neutralization of voltage sensor reduces gating charge12-16 charges/channel (3-4/subunit)
17how does voltage sensor move? if membrane potential concentrated across shallowpart of the protein,does not need to move far(Bezanilla, 2008)S4 (blue) rotates, pulling S6 (magenta) to open pore
18inactivation inactivation not required to restore resting Vm --what is inactivation good for?channels inactivate with a characteristic delaywhat controls inactivation?
19proteolysis reduces fast inactivation (N-type) (Hoshi et al, 1990; Zagotta et al, 1990)
20inactivation is not the opposite of activation reactivation requires two transitionsit is a distinct process triggered by conformational changes--characteristic delay encoded by protein
26AMPA receptor dimer of dimers upright teepee glutamate binds at D1-D2 interface,within monomer(Sobolevsky et al., 2009)
27how does glutamate binding open the channel? crystal structures of soluble domain suggest domain closure by ligand--pulls pore-lining helices aparthow can receptors respond to high-frequency release?
28inactivation is one method to terminate signaling (Sun et al, 2002)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
29desensitizationweakened D1-D1 interface relieves strain on D2, allowing channel closure
30distinguish signal from noise? how can a neurondistinguish signal from noise?--short EPSPs(excitatory postsynaptic potentials)require summation to reachthreshold, trigger action potential10 pA2 s
31K+ most channels conduct equally in both directions outinK+but some conduct more in one directionthan the other--rectification
32K+ inwardly rectifying K+ channels --K+ enters much faster than leaves outinwardly rectifying K+ channels--K+ enters much faster than leavesK+in(Bichet et al, 2003)returns Vm to EK for small depolarizationinactivates for large depolarizations--how?
33cytoplasm contains factors that promote rectification (Vandenberg, 1987)cytoplasm contains factors that promote rectification
34K+ 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
35several kinds of inward rectifiers IRKsKATPG protein-coupled (GIRKs)after depolarization inactivates IRK, all-or-none responsepositive feedback: voltage-activated Na+ or Ca++ channelsamplify the response, making it switch-likenegative feedback: voltage-gated K+ channelshyperpolarization-activated channels (Na+, Ca++)--contribute to oscillatory behavior
36membrane potentialion channelspermeationgatingConclusions1) Membrane potential (Vm) is determined by ionic gradients andthe relative permeability of different ions2) Very few ions need to flow to change membrane potential3) Channels drive Vm to the equilibrium potential of their permeant ions4) Permeability is determined by relatively weak interactionsand mutual repulsion of ions in the pore5) Channels can be gated by extracellular and intracellular ligandsand by membrane potential itself6) Inactivation (including desensitization) involves mechanismsdistinct from activation--for precise timing7) Channels can rectify, improving signal/noise8) Channels can be used to process information (switch-likecooperative responses, coincidence detection, oscillators)within single cells
37what do the changes in Vm accomplish? 1) propagate signal down axon2) trigger transmitter release at terminal3) activate contractile apparatus in muscle4) activate signaling proteins (e.g., kinases)5) regulate gene expressionbut how can changes in Vm do this if bulk concentrationsof 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 µM6) Vm also drives conformational changes in other membrane proteins--transporters (in what was probably its original role)
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