what is membrane potential? membrane potential is the charge difference across a membrane at the...
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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
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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
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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-
-+
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in
out
inK+
K+
+
-
membrane equivalent circuit
in the cell
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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)?
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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
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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)
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membrane potential
ion channels permeation gating
selectivity
(Doyle et al, 1998)
KcsA (bacterial K+ channel)
tetramer with 2 TMD/subunitinverted teepee
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hydration of ion replaced by backbone carbonyls --less effective for smaller Na+
cation does not interact with charged residues--why?
recognition of dehydrated ion
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Na+ channel
smaller ionbigger poreNa+ permeates semi-hydrated
(Payandeh et al., 2011)
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gating
membrane potential
ion channels permeation gating
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(TC Sudhof, 2008)
acti
on
po
ten
tial
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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)
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voltage sensor
K+ channel
Na+ channel
(Bezanilla, 2008)
basic residues aligned along one helixmovement of voltage sensor can be measured directly
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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
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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
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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)
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inactivation
inactivation not required to restore resting Vm --what is inactivation good for?
channels inactivate with a characteristic delaywhat controls inactivation?
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proteolysis reduces fast inactivation (N-type)
(Hoshi et al, 1990; Zagotta et al, 1990)
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inactivation is not the opposite of activationreactivation requires two transitionsit is a distinct process triggered by conformational changes --characteristic delay encoded by protein
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(TC Sudhof, 2008)
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steep dependence on [Ca++]o (synaptotagmin)nano-domain coupling:
(Eggermann et al., 2011)
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mechanism
wt
RIM DKO
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tighter coupling = enhanced synchrony
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(TC Sudhof, 2008)
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dimer of dimers
upright teepee
glutamate binds at D1-D2 interface, within monomer
(Sobolevsky et al., 2009)
AMPA receptor
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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?
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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)
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desensitization
weakened D1-D1 interface relieves strain on D2, allowing channel closure
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how can a neurondistinguish signal from noise?
--short EPSPs(excitatory postsynaptic potentials)require summation to reachthreshold, trigger action potential
10 pA2 s
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most channels conduct equally in both directions
out
in
but some conduct more in one directionthan the other--rectification
K+
K+
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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+
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cytoplasm contains factors that promote rectification
(Vandenberg, 1987)
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(Tao et al., 2009)
Rb+ (~K+) Sr++ (~Mg++)
K+ cannot displace Mg++, only with K+ efflux--rectification
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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)
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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
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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)
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Channels
Molecular Biology of the Cell, Chapter 11.Hille, B. Ion Channels of Excitable Membranes. Sinauer.
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