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
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