1. Membrane Potential and Ion Channels
Colin Nichols
Membrane Potential and Ion Channels
Background readings: Lodish et al., Molecular Cell Biology, 4th ed, chapter 15 (p ) and chapter 21 (p ). Alberts et al., Molecular Biology of the Cell, 4th ed, chapter 11 (p ) and p Hille Ionic channels of excitable membranes 3rd ed.

2. The Lipid Bilayer is a Selective Barrier
inside
outside
hydrophobic molecules (anesthetics)
gases (O2, CO2)
small uncharged polar molecules
large uncharged polar molecules
Ions
charged polar molecules (amino acids)
water

3. Ion Channel Diversity

4. Structure of a Potassium Channel
Doyle et al., 1998

5. Ion selectivity filter in KcsA
Doyle et al., 1998, Zhou et al., 2002

6. Ion selectivity filter in KcsA
Doyle et al., 1998, Zhou et al., 2002
Because of its smaller ionic radius, Na+ ions cannot bind pore regions

7. Ion selectivity filter in Na channels
(Also involves specific size cavity within larger molecule)
NavMs, NavAb, Kv1.2
Nature Communications , Oct 2, Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing Emily C McCusker,1, 3, Claire Bagnéris,1 Claire E Naylor,1, Ambrose R Cole,1 Nazzareno D'Avanzo,2, 4 , Colin G Nichols2, & B A Wallace1

8. Ion gradients and membrane potential
The inside of the cell is at ~ -0.1V with respect to the outside solution
-89 mV

9. Ion gradients and membrane potentialK Na Cl K
-89 mV

10. Ion gradients and membrane potential
Na 117
K 3
Cl 120
Anions 0
Total 240 Na K Cl
Anions 116
Total
[+ charge] = [- charge]
0 mV
[+ charge] = [- charge]
-89 mV

11. Movement of Individual K+ ions-
Na 117
K 3
Cl 120
Anions 0
Total 240
Na 30
K 90
Cl 4
Anions 116
Total 240 + -
[+ charge] = [- charge]
0 mV
[+ charge] = [- charge]
0 mV

12. Movement of Individual Cl- ions+ -
Na 117
K 3
Cl 120
Anions 0
Total 240
Na 30
K 90
Cl 4
Anions 116
Total 240
[+ charge] = [- charge]
0 mV
[+ charge] = [- charge]
0 mV
-89 mV

13. Capacitance Capacitance C Coulombs / Volt Farads F C = Q / V Q = C * V
I = dQ / dt = C * dV / dt
C µ area {
C µ 1 / thickness V C + - I
For biological membranes:
Specific Capacitance = 1 µF / cm2

14. - The Nernst Equation
Calculates the membrane potential at which an ion will be in electrochemical equilibrium.
At this potential: total energy inside = total energy outside
Electrical Energy Term: zFV
Chemical Energy Term: RT.ln[Ion]
Z is the charge, 1 for Na+ and K+, 2 for Ca2+ and Mg2+, -1 for Cl-
F is Faraday’s Constant = x 104 Coulombs / mole
R is the gas constant = Joules / °Kelvin * mole
T is the temperature in °Kelvin

15. At Electrochemical Equilibrium:
The concentration gradient for the ion is exactly balanced by the electrical gradient
There is no net flux of the ion
There is no requirement for any energy-driven pump to maintain the concentration gradient

16. Ion Concentrations Na 117 K 3 Cl 120 Anions 0 Total 240 Na 30 K 90
[+ charge] = [- charge]
0 mV
[+ charge] = [- charge]
-89 mV

17. Deviation from the Nernst Equation
Resting membrane potentials in real cells deviate from the Nernst equation, particularly at low external potassium concentrations.
The Goldman, Hodgkin, Katz equation provides a better description of membrane potential as a function of potassium concentration in cells.
Squid Axon
Curtis and Cole, 1942
-25
PK : PNa : PCl
1 : 0.04 : 0.05
-50
Resting Potential (mV)
-75
EK (Nernst)
-100
External [K] (mM)

18. The Goldman Hodgkin Katz Equation
Resting Vm depends on the concentration gradients and on the relative permeabilities to Na, K and Cl. The Nernst Potential for an ion does not depend on membrane permeability to that ion.
The GHK equation describes a steady-state condition, not electrochemical equilibrium.
There is net flux of individual ions, but no net charge movement.
The cell must supply energy to maintain its ionic gradients.

19. Summary:
Cell membranes form an insulating barrier that acts like a parallel plate capacitor (1 µF /cm2)
Ion channels allow cells to regulate their volume and to generate membrane potentials
Only a small number of ions must cross the membrane to create a significant voltage difference
~ bulk neutrality of internal and external solution
Permeable ions move toward electrochemical equilibrium
Eion = (60 mV / z) * log ([Ion]out / [Ion]in) @ 30°C
Electrochemical equilibrium does not depend on permeability, only on the concentration gradient

20. Summary (continued):
The Goldman, Hodgkin, Katz equation gives the steady-state membrane potential when Na, K and Cl are permeable
In this case, Vm does depend on the relative permeability to each ion and there is steady flux of Na and K
The cell must supply energy to maintain its ionic gradients

21. Voltage-Gated Channels and Action Potentials
Colin Nichols
Voltage-Gated Channels and Action Potentials

22. The Variety of Action Potentials
Skeletal muscle
Cardiac muscle

23. The action potential – Physics in cell biology1 2 3 4 K+
Na+

24. Currents During an Action Potential
Time Course of Currents

25. Sodium Channel Gating States

26. AChR – non-selective cation channel
(Permeable to Na+ and K+)

27. Passive vs. Active
All cells exhibit passive changes in membrane potential when stimulated
Only excitable cells fire action potentials
Excitability depends on specialized channels

28. Voltage sensing – structural basis?

29. Voltage sensing – more than just channels!
Ciona intestinalis voltage-sensor containing phosphatase, Ci-VSP, which couples a VSD to a phosphatase and tensin homolog (PTEN)-like domain. Murata et al. Nature 435, 1239–1243 (2005).

30. Voltage dependent inactivation – structural basis?
“Ball and chain inactivation”

31. Summary: Action potentials require voltage-gated channels
They open with depolarization and carry a net inward current
Inactivation and / or voltage-gated outward current underlie repolarization
Inactivation imposes a refractory period
The final effect of an action potential is to elevate calcium
Channel structure gives insight into gating and permeation

32. Channelopathies Or… How can a single amino acid change in an ion channel protein lead to a whole disease syndrome?

33. The islet of Langerhans

34. Glucose-dependent b-cell electrical activity

35. Central role of KATP channel in control of insulin secretion
Normal b-cell response to a rise in blood glucose
KATP channel is inhibited, turning on insulin secretion in pancreatic b-cells…?
Glucose
Glucose
Glut2
Glucose
Blood
Insulin
ATP
ADP - Ca 2+ +
KATP

36. Hyperinsulinemia in absence of KATP channel activity ?
b-cell response to a fall in blood glucose in absence of KATP
KATP channel is not activated, insulin secretion is maintained, and blood glucose drops further…?
Glucose
Glut2
Glucose
Blood
Insulin
ATP
ADP Ca 2+ +
KATP

37. Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI)
A rare genetic disease characterized by high insulin in parallel with low glucose.
This disease affects newborn infants - 1 case for 40,000 people is present, but can rise to 1:2500 in regions of high consaguinity.
Serious complication is brain damage.
Treatment objective is to maintain normal blood sugar - using [1] medication (octreotide and/or diazoxide) to inhibit insulin secretion, or [2] in cases where there is no response to drugs -surgical removal of the pancreas becomes necessary.
Even in non-pancreatectomized individuals, PHHI can progress to Type II diabetes - Huopio et al., 2003
The genes identifed at 11p15.1as responsible for at least 50% of cases of PHHI are the high affinity sulfonylurea receptor (SUR1) and Kir6.2.

38. Central role of KATP channel in control of insulin secretion
Normal b-cell response to a rise in blood glucose
KATP channel is inhibited, turning on insulin secretion in pancreatic b-cells…?
Glucose
Glucose
Glut2
Glucose
Blood
Insulin
ATP
ADP - Ca 2+ +
KATP

39. Diabetes if KATP channels become overactive?
If KATP channel is not inhibited, insulin secretion remains turned off?
Glucose
Glucose
Glut2
Glucose
Blood
Insulin
ATP
ADP Ca 2+
KATP

40. ATP-insensitivity of KATP channels as a causal mechanism of neonatal human diabetes?
Human neonatal diabetes is characterized by no insulin secretion in parallel with high glucose. Fasted blood glucose >> 15 mM, ketoacidosis frequently present, and low birth weights are reported.
Detected from birth to ~1 year old. Maybe
permanent (PNDM) or transient (TNDM). Most
severe syndromic PNDM is associated with
neurological features and muscle weakness.
Assumed to be early Type 1 Diabetes –Treatment
objective is to maintain normal blood sugar - using
insulin injections.