membrane potential and ion channels colin nichols background readings: lodish et al., molecular cell...

Membrane Potential

and Ion Channels

Colin Nichols

Background readings:Lodish et al., Molecular Cell Biology, 4th ed, chapter 15 (p. 633-665) and chapter 21 (p. 925-965).

Alberts et al., Molecular Biology of the Cell, 4th ed, chapter 11 (p. 615-650) and p. 779-780.

Hille Ionic channels of excitable membranes 3rd ed.

LIFE – A complex interplay of chemistry and physics

The action potential – Physics in cell biology

1

1

2

2

3

3

4

4

K+

Na+

Excitation-contraction coupling

The islet of Langerhans

Glucose-dependent hormone secretion

The Lipid Bilayer is a Selective Barrier

hydrophobic molecules (anesthetics)

gases (O2, CO2)

small uncharged polar molecules

large uncharged polar molecules

Ions

charged polar molecules (amino acids)

water

inside outside

Ion Channel Diversity

Cation channel Subunit Topology

Cation channel Subunit Topology

The ‘inner core’ of cation channel superfamily members

Structure of a Potassium Channel

Doyle et al., 1998

Selectivity Filter and Space-Filling Model

Doyle et al., 1998

Ion selectivity filter in KcsADoyle et al., 1998, Zhou et al., 2002

Selectivity filter diversity

Ion selectivity filter in Na channels

NavMs, NavAb, Kv1.2 Nature Communications , Oct 2, 2012. 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

Views of the Channel Open and Shut

Jiang et al., 2002

Views of the Channel Open and Shut

Nature Communications , Oct 2, 2012. 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

Ion gradients and membrane potential

Ion gradients and membrane potential

Ion gradients and membrane potential

Ion gradients and membrane potential

-89 mV

How does this membrane potential come about?

The inside of the cell is at ~ -0.1V with respect to the outside solution

Ion gradients and membrane potential

-89 mV

How does this membrane potential come about?

K

Cl

K Na

Ion gradients and membrane potential

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ charge] = [- charge]

-89 mV

How does this membrane potential come about?

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ charge] = [- charge]

0 mV

+ -

+ -

Movement of Individual K+ ions

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ charge] = [- charge]

0 mV

Movement of Individual Cl- ions

+ -

+ -

-89 mV

Setting a membrane potential –questions!

How many ions must cross the membrane to set up this membrane potential?

What makes it stop at this potential?

Capacitance

Capacitance C Coulombs / Volt Farads F

C = Q / V Q = C * V I = dQ / dt = C * dV / dt

V C+

-

I

+ + + + + + + +

- - - - - - - -

C µ area

{

C µ 1 / thickness

For biological membranes:Specific Capacitance = 1 µF / cm2

How many ions?

How many ions must cross the membrane of a spherical cell 50 µm in diameter (r = 25 µm) to create a membrane potential of –89 mV?

Q (Coulombs) = C (Farads) * V (Volts)

Specific Capacitance = 1.0 µFarad / cm2

Surface area = 4 r2

Faraday’s Constant = 9.648 x 104 Coulombs / mole

Avogadro’s # = 6.022 x 1023 ions / mole

Calculations

Area = 4 r2

= 4 p (25 x 10-4 cm)2

= 7.85 x 10-5 cm2

= 78.5 x 10-6 µFarads

= 78.5 x 10-12 Farads

Q = C * V

= 78.5 x 10-12 Farads * 0.089 Volts

= 7 x 10-12 Coulombs

~ 7 x 10-12 Coulombs / 9.65 x 104 Coulombs per mole

= 7.3 x 10-17 moles of ions must cross the membrane

= 0.073 femptomoles or ~ 44 x 106 ions ~ 1.1 µM change in [ ]

1 2

3

Why does it stop? - 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: zFVChemical 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 = 9.648 x 104 Coulombs / moleR is the gas constant = 8.315 Joules / °Kelvin * moleT is the temperature in °Kelvin

Nernst Equation Derivation

zF.Vin + RT.ln [K+]in = zF.Vout + RT.ln [K+]out

zF (Vin – Vout) = RT (ln [K+]out – ln [K+]in)

EK = Vin – Vout

= (RT/zF).ln([K+]out/[K+]in)

EK = 2.303 (RT / F) * log10([K+]out/[K+]in)

In General:

Eion ~ 60 * log ([ion]out / [ion]in) for monovalent cation at 30°

Nernst Potential Calculations

First K and ClEK = 60 mV log (3 / 90) = 60 * -1.477 = -89 mV

ECl = (60 mV / -1) log (120 / 4) = -60 * 1.477 = -89 mV

Both Cl and K are at electrochemical equilibrium at -89 mV

Now for Sodium ENa = 60 mV log (117 / 30) = 60 * 0.591 = +36 mV

When Vm = -89 mV, both the concentration gradient and electrical gradient for Na are from outside to inside

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

Ion Concentrations

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ charge] = [- charge]

-89 mV

Ion Concentrations

Na 117K 3Cl 120Anions 0Total 240

Na 30K 90Cl 4Anions 116Total 240

[+ charge] = [- charge]

0 mV

[+ charge] = [- charge]

-89 mV

Add water – 50% dilution

Na 30K 90Cl 4Anions 116Total 240

Environmental Changes: Dilution

Na 58.5K 1.5Cl 60Anions 0Total 120

[+ charge] = [- charge]

Add water – 50% dilution

[+ charge] = [- charge]

-89 mV

H2O

Na 30K 90Cl 4Anions 116Total 240

Environmental Changes: Dilution

Na 58.5K 1.5Cl 60Anions 0Total 120

[+ charge] = [- charge]

Add water – 50% dilution

EK = -107 mVECl = -71 mV

[+ charge] = [- charge]

-89 mV

Environmental Changes: Dilution

Na 58.5K 1.5Cl 60Anions 0Total 120

[+ charge] = [- charge]

Add water – 50% dilution

EK = -107 mVECl = -71 mV

Na 30K <90Cl <4Anions 116Total <240

[+ charge] = [- charge]

-89 mV

H2O

Deviation from the Nernst Equation

1 3 10 30 100 300 External [K] (mM)

0

-25

-50

-75

-100

Res

ting

Pot

entia

l (m

V)

EK (Nernst)

PK : PNa : PCl

1 : 0.04 : 0.05

Resting membrane potentials in real cells deviate from the Nernst equation, particularlyat low external potassiumconcentrations.

The Goldman, Hodgkin, Katzequation provides a better description of membrane potential as a function of potassium concentration in cells.

Squid AxonCurtis and Cole, 1942

The Goldman Hodgkin Katz Equation

outClinNainK

inCloutNaoutKm [Cl]P[Na]P[K]P

[Cl]P[Na]P[K]Plog60mVV

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.

GHK Equation: Sample Calculation

120390

47.113log60mVVm

= 60 mV * log (18.7 / 213)

= 60 mV * -1.06

= -63 mV

Suppose PK : PNa : PCl = 1 : 0.1 : 1

Recording Membrane Potential

To here L1

On-Cell Patch Recording (Muscle)

50 µm

Forming a Tight Seal

Whole-Cell and Outside-Out Patch

10 µm

Channels Exist in at least 2 States

To here…

Current ~3.2 pA = 3.2 x 10-12 Coulombs/sec

Charge on 1 ion ~1.6 x 10-19 Culombs

Ions per second = 3.2/1.6 x 10^7

~20 million

Exponential Distribution of Lifetimes

mean open time = open = 1 / a = 1 / closing rate constant

Summary:

I. Cell membranes form an insulating barrier that acts like a parallel plate capacitor (1 µF /cm2)

II. Ion channels allow cells to regulate their volume and to generate membrane potentials

III. Only a small number of ions must cross the membrane to create a significant voltage difference~ bulk neutrality of internal and external solution

IV. 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

Summary (continued):

V. 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

outClinNainK

inCloutNaoutKm [Cl]P[Na]P[K]P

[Cl]P[Na]P[K]Plog60mVV