1. Electrophysiology 1

2. Beginning electrophysiology: How the resting potential is generated
How do we know there’s a resting potential?
Its origins: ionic concentrations inside and outside cells
Ion pumps and ion channels in the membrane
K+ ions and the Nernst equation
Na+ ions and the Goldman-Hodgkin-Katz equation
The role of the Na+/K+ ATPase 2

3. The resting potential How do we know there’s a resting potential?
How was it first measured? 3

4. Resting potential first measured by Bernstein (1902)
Electrode touching intact muscle
(i.e. extracellular)
Electrode touching cut end
(i.e. intracellular)
Cut end of muscle
Muscle (cut in the middle)
Oil 4

5. More modern approaches
Squid giant axon 5

6. More modern approaches
A real squid giant axon
It’s so big you can push a wire along it! 6

7. More modern approaches
Methods for smaller neurones 7

8. So how can biological tissue generate electricity?
Our body fluids contain ions
A solution of ions is electrically neutral: equal numbers of (+) and (–) ions
Potential difference can be created by separating (+) and (–) ions
Cells have a membrane: that’s where the separation happens (membrane lets some charges through and not others)
(+) and (–) ions are separated there creating a voltage difference across the cell membrane (i.e. a membrane potential) 8

9. Movement of ions across the membrane
At rest: Charge separation due to membrane:
more (–) inside
more (+) outside
During action potential:
(+) charges move inside
leaving excess (–) outside 9

10. Evidence for importance of the membrane (1)
Advance microelectrode slowly into cell
This is what happens 10

11. Evidence for importance of the membrane (2)
Squid axon
Take away everything apart from the membrane!
This is what happens 11

12. Closer look at the cell membrane
Lipid bilayer
Protein molecules 12

13. The cytoskeleton supports the cell membrane
A simple “fluid mosaic” membrane would have little mechanical strength
Cytoskeletal proteins e.g. actin, spectrin, ankyrin support the bilayer and attach proteins 13

14. How do substances move across the membrane?14

15. - two types of proteins in the membrane
How can ions cross it?
- two types of proteins in the membrane 15

16. The resting potential
How is the resting potential generated? 16

17. Bernstein and the resting potential
This is what was known in 1902 about ions in biological tissue
–85 mV
Proteins: large anions 17

18. Bernstein and the resting potential
To create a voltage difference, the membrane has to let some ions cross and stop others from crossing
This happens if only one type of ion channel is open
Let’s see what would happen if only K+ channels are open 18

19. Bernstein and the resting potential
Begin with no resting potential
K+ ions would start to move randomly
More would move outwards than inwards
(because there are more on the inside) 19

20. Bernstein and the resting potential
Inside would become negative
Outward flow would decrease, inward flow increase
+ – 20

21. Bernstein and the resting potential
Inside would become negative
Outward flow would decrease, inward flow increase
+ – 21

22. Bernstein and the resting potential
Inside would become negative
Outward flow would decrease, inward flow increase
The inside would become still more negative
+ –
+ – 22

23. Bernstein and the resting potential
+ –
+ – 23

24. Bernstein and the resting potential
Process would continue till inward and outward flows are the same: equilibrium
Movement wouldn’t stop: it would be equal and opposite
–85 mV
This could explain the resting potential
+ –
+ –
+ – 24

25. How to test this?
If we change the outside K+ concentration the potential ought to change
+ –
–85 mV
We can predict exactly how it ought to change: if [K+]o = [K+]i, then Em should be zero
30 Na+ K+
0 mV 25

26. Effect of [K+]o on the resting potential
Measured in squid axon (using wire pushed down axon) 26

27. Effect of [K+]o on the resting potential
Squid axon: [K+]i = ~400 mM
So raising [K+]o does bring resting potential nearer to zero
What about a more quantitative prediction? 27

28. The Nernst equation
Predicts the voltage that would result from different ion concentrations
Looks like this:
Em: membrane potential
R: the gas constant (8.315 J mol–1 K–1)
T: absolute temperature (20 °C = 293 K)
z: charge on the ion (z=1 for K+)
F: Faraday’s constant (96480 C mol–1)
[K+]o, [K+]o: K+ ion concentrations outside and inside cell
ln: natural logarithm
Why does it look like this?
If you want to follow this up, see the derivation on Blackboard 28

29. What does the Nernst equation predict?
Prediction 1: If [K+]o < [K+]i then the inside will be negative
Prediction 2: If [K+]i = [K+]o then Em = 0 mV
Prediction 3: The more similar [K+]i and [K+]o are, the smaller is Em; so raising [K+]o at constant [K+]i will depolarise 29

30. Does the Nernst equation predict resting potential correctly?
Back to our earlier measurements
Now we plot these: resting membrane potential against [K+]o 30

31. Resting potential and [K+]o31

32. Resting potential and [K+]o
Nernst equation is good at high [K+]o, but not at low [K+]o
How to account for this?
The membrane is also permeable to Na+ 32

33. Effect of the sodium permeability
+ –
–85 mV
+ –
–65 mV
More Na+ enters than leaves
because of the concentration gradient and the inside negativity
The cell becomes less negative inside 33

34. The Nernst equation with Na+ permeability
becomes the Goldman-Hodgkin-Katz (GHK) equation:
...how can we picture this in physical terms? 34

35. What does the GHK equation mean?
+ –
–65 mV
Goldman-Hodgkin-Katz (GHK) equation:
Inward fluxes
Outward fluxes 35

36. Does this account for the deviation?
GHK prediction
Deviation from Nernst prediction at low [K+]o
is accounted for well by permeability to Na+
(The GHK equation is a good fit) 36

37. It’s no longer equilibrium...
+ –
–65 mV
+ –
–85 mV
There is a net gain of Na+
and a net loss of K+
So how does the cell avoid running down? 37

38. Ionic pumping Passive fluxes of K+ (out) and Na+ (in)
are balanced by the Na+/K+ ATPase
It pumps Na+ out and K+ in
This keeps ionic concentrations stable
Passive ionic fluxes
Active pumping 38

39. Ionic pumping is electrogenic (i.e. it changes membrane potential)
3 Na+
2 K+
Two K+ ions move in but 3 Na+ ions go out:
So the pumping creates a current
(+) charge moves out:
Inside becomes more negative
This makes resting potential more negative than GHK prediction
Na+/K+ ATPase 39

40. How ionic pumping affects resting potential?
Recorded from a mollusc neurone
4 °C: Na+/K+ ATPase is inactive: GHK prediction fits well
17 °C: Na+/K+ ATPase is active: deviation from GHK prediction - membrane potential is more negative 40

41. Summary: three determinants of resting potential
Major role for K+ ions which is described by the Nernst equation
This describes a true equilibrium
Deviation from Nernst prediction due to Na+ permeability
Makes resting potential less negative
Described by Goldman-Hodgkin-Katz equation
Non-equilibrium: the cell would run down were it not for the Na+/K+ ATPase
The Na+/K+ ATPase pumps more Na+ out than K+ in: makes resting potential more negative 41

42. Reading for today’s lectures:
Purves et al chapter 2
Further reading:
Nicholls et al chapter 5
Kandel et al chapter 7
Next lecture: The action potential
Reading for next lecture:
Purves et al chapter 2 (later part on action potential)
Further reading:
Nicholls et al pages 26-31, 62-63, 91-93
Kandel et al chapter 8 42