Ion Channel Function

Measuring Currents

Function of ion channels:

measuring ionic currents
What is a current?
Voltage clamp
Single channel recording
The case of equal ion concentration
The case of a concentration gradient
I/V plots
Macroscopic currents
Probability of ion channel opening
I/V plots: ohmic and non ohmic (rectifying channels)
Reversal potential of multi ion permeable channels.

Tuesday, February 2, 16
Definition of Current: net flow of charge.
By convention, direction of current flow= direction of flow of
positive charge.

Conditions for getting current flow across membrane?

1. Something has to carry the charge (there must be ions)

2. Have to have a passageway (channels need to be open)

3. Must have a driving force (concentration gradient, voltage)

Hodgkin and Huxley :

Measured the currents during an action potential
in squid giant axons.
Current Clamp Voltage Clamp

They predicted existence of ion channels

which carry these currents

Tuesday, February 2, 16
 Patch-clamp method allows
recording of current from
single channels - can detect
conformational changes of a
single protein!

Tuesday, February 2, 16
 The Patch Clamp Method - YouTube

Neuroscience 5e: Chapter 4 Animations

Voltage Clamp

Current

Vc = Command Voltage

The current injected by the amplifier is equal to but

opposite in sign to the current that is flowing
through the cell.

Tuesday, February 2, 16
 Voltage clamp on a single K channel

inside

outside

Outside-out patch

1. The case of no concentration gradient: [K]out=[K]in

+20 mV I Out
K+
0 mV I

K+
Fluctuations between open and closed states

1. The case of no concentration gradient: [K]out=[K]in

+20 mV I Out
K+
0 mV I

-20 mV I In
K+

-40 mV I
I/V plot
I current

Out *
V membrane Current flow through
-40 -20 * +20 Potential channel obeys Ohms
In law: I = gV
*
*
Tuesday, February 2, 16
 Ohms Law

V = Voltage (volts - typically mV)

V=IR I =Current (amperes - typically pA)
R =Resistance (ohms - typically M)
g =Conductance (siemens - typically pS)

R=1/g
V=I/g
VI

What determines the slope?

I
V=IR
Out
V I/V=1/R
I/V=g
In
gI
Conductance is a combined
measure
of permeability and number of ions
[ion] g slope

Ohmic relationship

Tuesday, February 2, 16
 2. The case of a concentration gradient: [K]in > [K]out
In

K+ 0 mV I out
Out
K+

When not in voltage clamp

K+
K+ = K+ No net flow of K (equilibrium)
+

+
+ +- - +
- + -- +
K Nernst Equation Eion= RT ln [K]out
ZF [K]in
Equilibrium potential (Eion) is voltage at
which driving force due to concentration Eion=58 log [K]out
gradient and driving force due to voltage
exactly balance one another [K]in

Nernst Equation EK=58 log [K]out

[K]in

[K]in =100mM Log 1/10=-1

[K]out =10mM
EK=-58 mV

K+

+
+ +- - +
- + -- +
K

Tuesday, February 2, 16
 EK=-58 mV
In +20 mV I
K+ 0 mV I Out
Out
K+
-58 mV I

-70 mV I In

I current
*
Out
V membrane
-70
*
-58 +20 Potential
In
*

???
Tuesday, February 2, 16
 The Neuro 102 Puzzler !!
If the concentra,on of sodium (Na) outside the cell is
10 ,mes greater than inside the cell, what will the IV plot
of a single Na channel look like?

Nernst Equation ENa=58 log [Na]out

[Na]in
[Na]in =10mM log 10=1
[Na]out =100mM ENa=58 mV

In

Na+
+70 mV I Out
Out +58 mV I
Na+ 0 mV I In

-20 mV I

Tuesday, February 2, 16
 Single Na channel I/V plot

I current

Out
V membrane
* Potential
In +58

Single channel currents

i current
K+ Na+ [Na+]out > [Na+]in

Out
V membrane
Poten0al
In

Reversal Potential (Eion)

V = IR i = single channel current
i=Vg g = single channel conductance
V is not the absolute membrane poten,al but rather the
dierence between the membrane poten,al and the equilibrium
poten,al.
i=(Vm-E ion)g
Magnitude of the current depends on ion concentra,on gradient,
membrane poten,al and the conductance
Tuesday, February 2, 16
 i current
K+ V = IR
current i=Vg
Out
V membrane
Potential
In
Driving force
Vm
E ion

i=(Vm-Eion)g
V is not the absolute membrane potential but rather the
difference between the membrane potential and the equilibrium
potential.

I current
K+ i=(Vm-Eion)g

Out
V membrane
Potential
In

1. Increase ion concentration, but

keep relative ratio constant

2. Alter concentration gradient

How does ion concentration affect I/V relation?

Tuesday, February 2, 16
 Another Neuro 102 Puzzler!

What would a single channel I/V plot

for Cl- channel look like?

Tuesday, February 2, 16
 Microscopic and Macroscopic Currents

Outward

Many single channel currents

summate to create the whole cell
(macroscopic) current.
Inward

Na+ channel
K+ channel
V
V
ipA i

I nA I nA
Short delay in opening, inactivate Delay in opening, dont inactivate

Tuesday, February 2, 16
 Single channel currents build the whole cell (macroscopic
current).
I = macroscopic current
i = single channel current (micro)
I = i . n . Po n = # channels
Po = probability of open channel

Po is dynamic - regulation is due to

gating and modulation

What is channel gating?

Transition of channels between open and closed states.

Voltage
ligand Single channel recording
temperature
light
stretch

C O I
Closed open Inactivated

Activation curve

1-
Open In presence of a stimulus the
probability
probability of open state is increased

Vm

Tuesday, February 2, 16
 Gating of ion channels

Voltage dependence of ion channels affects macroscopic I.

Need to determine the probability of channel

opening as a function of voltage.

Activation Curve
Probability of At 0 mV the channel is
Channel opening 1-
open half of the time.

Vm

K+ and Na + activation curves are very similar

What does an IV plot of macroscopic current look like?

Non voltage gated channel
pA i P open
1

A I
-50 +50 V -50 +50 V (mV)

-50 +50 V I=i . n . Po

P open
pA Voltage gated channel
i 1

A I Linear

-50 +50 V -50 +50 V (mV)

Exponential
-50 +50 V Difference in both magnitude
and voltage dependence

What would the macroscopic Na+ current look like?

Tuesday, February 2, 16
 Rectification: non ohmic IV relationship

Current passes in one direction better

than it does in another

i i

Vm Vm

Inward Rectifier Outward Rectifier

K inward rectifier : Open at rest, little outward current

i

Voltage dependent block

can be caused by
mV intracellular cations.

Voltage clamp while changing intracellular Mg++

[Mg++] (mM)
0
Some Kir channels i 0.2
still rectify with no
internal Mg.
3
They are blocked by 10
Polyamines
(spermine). V
Lu and Mckinnon 1994

Tuesday, February 2, 16
 Measuring single channel currents of multi-ion permeable
channels and ligand gated channels

Nicotinic ACh receptor: Ligand gated channel (receptor)

In presence of ACh!
i +20 mV I
Out
0 mV I

Vm -20 mV I In
-40 mV I
Reversal Potential
(Vrev)

nACh receptor: permeable to both Na+ and K +

nACh receptor
+20 mV I
i Out
0 mV I
EK ENa
Vm
-20 mV I In
-40 mV I

Which ions flow at each voltage and in what direction?

Remember the current of individual ions will depend on the

driving force on that ion.
At EK the driving force on K is zero no K current
But large driving force on Na inward Na current.

At the nAChR reversal potential, I=0, net flow of charges is

zero, thus: I K+=I Na+

Tuesday, February 2, 16
 At Reversal Potential IK+=I Na+

To calculate the reversal potential of the nAChR need to take into

account both ENa+ and EK+ and relative permeability to the ions.

(gNa . ENa+ ) + (gK . EK+ )

V rev=
gNa + gK
nACh receptor
Goldman-Hodgkin-Katz Equation
i

Summary

V=IR
Voltage clamp- allows to measure currents at different
voltages
Single channel recordings.
IV plots
Reversal potential (for single ions= Nernst equilibrium)
Driving force (difference between Vm and Eion)
Macroscopic currents (I = i . n . PO)
IV plots of multi ion permeable channels.

Tuesday, February 2, 16
Tuesday, February 2, 16