1. Permeability changes during the action potential1

2. Studying voltage-gated channels
Starting point:
We suspect voltage is opening/closing the channels
Hence we have to hold voltage constant to provide a constant stimulus
For this we use the VOLTAGE CLAMP method 2

3. Voltage clamp method
Em more negative than command potential: Amplifier output goes positive thus making Em more positive
Em more positive than command potential: Amplifier output goes negative thus making Em more negative
So amplifier keeps Em at command potential
We measure the current it produces 3

4. So what do we see on depolarisation?
Late sustained outward current
Early transient inward current 4

5. Hyperpolarisation: almost no current5

6. What ions carry the current?
K+?
Na+?
...how can we test this?
“Ion substitution” 6

7. Na+ ions and the early transient current
i.e. current due to Na+ 7

8. Another approach to separating currents
Tetrodotoxin: toxin from Fugu puffer fish
Blocks Na+ channels 8

9. Tetrodotoxin used to separate currents
Total ionic current in human node of Ranvier
Current with tetrodotoxin
Difference current:
i.e. current through Na+ channels
Schwarz, Reid & Bostock 1995 9

10. Characteristics of Na+ and K+ currents
Slower activation than Na+ current
No inactivation on timescale of an AP
Na+ current:
Very fast activation
Fast inactivation 10

11. Studying single ion channels
To record single ion channels we need a tiny patch of membrane
We still have to control the voltage across the membrane
These combine to give us the PATCH CLAMP method 11

12. Recording single ion channel currents
Patch clamp recording
Cell attached recording 12

13. Recording single ion channel currents
Patch clamp recording
Excised patch recording (“inside-out”) 13

14. Recording single ion channel currents
Patch clamp recording:
how the currents are recorded
Current
Membrane patch 14

15. Na+ channel activation and inactivation
-60 mV
Voltage (Em)
Current
–90 mV
closed
open
Depolarisation opens the channel:
activation
It closes again spontaneously:
inactivation
Reid et al 1991 15

16. Activation and inactivation of a Na+ channel
+ + +
– – – 16

17. Activation and inactivation of a Na+ channel
+ + +
– – –
– – –
+ + + 17

18. Activation and inactivation of a Na+ channel
+ + +
– – –
– – –
+ + + 18

19. Activation and inactivation of a Na+ channel
+ + +
– – –
– – –
+ + + 19

20. Activation and inactivation of a Na+ channel
+ + +
– – –
– – –
+ + + 20

21. Activation and inactivation of a Na+ channel
+ + +
– – –
+ + +
– – –
– – –
+ + + 21

22. Activation and inactivation of a Na+ channel
+ + +
– – –
+ + +
– – – 22

23. Activation and inactivation of a Na+ channel
+ + +
– – – 23

24. Ion channels are proteins
Lipid bilayer
Protein molecules 24

25. Ion channels are proteins
They are composed of amino acids
So their properties result from their amino acid sequence
How can we understand how structure determines function?
We need to know 3 things:
what is the amino acid sequence?
what is the 3-dimensional structure? (i.e. where are the amino acids?)
what happens when we change individual amino acid residues?
For some ion channels, we have the answers to these questions
Firstly, what is the amino acid sequence? 25

26. 1. What is the amino acid sequence?
Direct protein sequencing works only for short peptides
It’s easier to determine the messenger RNA sequence 26

27. mRNA and protein sequence
So:
If we know the mRNA sequence we can work out the amino acid sequence
Three bases in mRNA code for one amino acid in the protein 27

28. Working out the mRNA sequence
Beads with TTT attached: mRNA sticks
Wash off all the rest
Then separate mRNA from beads 28

29. Working out the mRNA sequence29

30. Working out the mRNA sequence30

31. Working out the mRNA sequence31

32. Expressing ion channels
mRNA
Oocytes 32

33. Working out the mRNA sequence
But what do we screen for?
We need a starting sequence
Example:
- K+ channel from Drosophila (fruit fly) 33

34. Drosophila K+ channel Starting point: Shaker mutant flies
Physiological evidence: Shaker is a K+ channel mutation
Chromosomal location of mutation known: position 16F on X chromosome 34

35. Drosophila K+ channel Starting point: Shaker mutant flies
Physiological evidence: Shaker is a K+ channel mutation
Chromosomal location of mutation known: position 16F on X chromosome
Starting with a chromosomal DNA clone known to be from 16F...
Probe a library of chromosomal DNA to find overlapping clones...
Then use these clones to further probe the library
Method known as CHROMOSOME WALK 35

36. Drosophila K+ channel
Region 16F on X chromosome
Starting clone
Overlapping clone 1
Probe with end only
Overlapping clone 2
Probe with end only
Overlapping clone 3
etc
etc
etc
Now we know the chromosomal DNA sequence (exons and introns) but not the mRNA sequence (exons only)
Next step: positive clones used to probe cDNA library 36

37. Drosophila Shaker K+ channel: mRNA sequence
>gi|157063|gb|M |DROCHAB D.melanogaster potassium channel (Shaker) mRNA, complete cds
GAATTCCGGAGTTTCTATCCAGACTTCAATATTTTTTTACCTCGCTCAAAACCCCCCACTCGCACTTTAAATAATAAAAAAAAGCAGGTGGTGCGTGCCGCGTAGCCGCGCGTGATTCTTGTTGTTGTTTTTTTTTTTTCGGTGAATCTCTTGTAACCATGTACCAAAGTTCTTTGCCGCGAAAACTAAAATGAAAACGAAAGTGAAAATGAGCGAATGGCAGCCGCGGCCACAGCAATCGATCCATGACACAACCAGTGACAAGCAGTCCCCCAGTGAAACCGCATCCGCATCCGAGTCCGATACCGATAAAGATTCTGAATCGGAGTGAGTGCCGCGTCCGAGAGCGTTCCCTGTCCACGTCCACCATCGGCGGAGCAGGTGTGCCTGAGGCCCACCTGGTGGCATGGCCGCCGTTGCCGGCCTCTATGGCCTTGGGGAGGATCGCCAGCACCGCAAGAAGCAGCAGCAACAGCAGCAGCACCAGAAGGAGCAGCTCGAGCAGAAGGAGGAGCAAAAGAAGATCGCCGAGCGGAAGCTGCAGCTGCGGGAGCAGCAGCTCCAGCGCAACTCCCTCGATGGTTACGGGTCTTTGCCCAAATTGAGCAGTCAAGACGAAGAAGGGGGGGCTGGTCATGGCTTTGGTGGCGGACCGCAACACTTTGAACCCATTCCTCACGATCATGATTTCTGCGAAAGAGTCGTTATAAATGTAAGCGGATTAAGGTTTGAGACACAACTACGTACGTTAAATCAATTCCCGGACACGCTGCTTGGGGATCCAGCTCGGAGATTACGGTACTTTGACCCGCTTAGAAATGAATATTTTTTTGACCGTAGTCGACCGAGCTTCGATGCGATTTTATACTATTATCAGAGTGGTGGCCGACTACGGAGACCGGTCAATGTCCCTTTAGACGTATTTAGTGAAGAAATAAAATTTTATGAATTAGGTGATCAAGCAATTAATAAATTCAGAGAGGATGAAGGCTTTATTAAAGAGGAAGAAAGACCATTACCGGATAATGAGAAACAGAGAAAAGTCTGGCTGCTCTTCGAGTATCCAGAAAGTTCGCAAGCCGCCAGAGTTGTAGCCATAATTAGTGTATTTGTTATATTGCTATCAATTGTTATATTTTGTCTAGAAACATTACCCGAATTTAAGCATTACAAGGTGTTCAATACAACAACAAATGGCACAAAAATCGAGGAAGACGAGGTGCCTGACATCACAGATCCTTTCTTCCTTATAGAAACGTTATGTATTATTTGGTTTACATTTGAACTAACTGTCAGGTTCCTCGCATGTCCGAACAAATTAAATTTCTGCAGGGATGTCATGAATGTTATCGACATAATCGCCATCATTCCGTACTTTATAACACTAGCGACTGTCGTTGCCGAAGAGGAGGATACGTTAAATCTTCCAAAAGCGCCAGTCAGTCCACAGGACAAGTCATCGAATCAGGCTATGTCCTTGGCAATATTACGAGTGATACGATTAGTTCGAGTATTTCGAATATTTAAGTTATCTAGGCATTCGAAGGGTTTACAAATATTAGGACGAACTCTGAAAGCCTCAATGCGGGAATTAGGTTTACTTATATTTTTCTTATTTATAGGCGTCGTACTCTTCTCATCGGCGGTTTATTTTGCGGAAGCTGGAAGCGAAAATTCCTTCTTCAAGTCCATACCCGATGCATTTTGGTGGGCGGTCGTTACCATGACCACCGTTGGATATGGTGACATGACGCCCGTCGGCTTCTGGGGCAAAATTGTCGGCTCTTTGTGCGTGGTCGCTGGTGTGCTGACAATCGCACTGCCGGTACCGGTTATCGTCAGTAATTTCAATTACTTCTATCACCGCGAAGCGGATCGGGAGGAGATGCAGAGCCAAAATTTCAACCACGTTACAAGTTGTTCATATTTACCTGGTGCACTAGGTCAACATTTGAAGAAATCCTCACTCTCCGAATCGTCGTCGGACATAATGGATTTGGATGATGGCATTGATGCAACCACGCCAGGTCTGACTGATCACACGGGCCGCCACATGGTGCCGTTTCTCAGGACACAGCAGTCATTCGAGAAGCAGCAGCTCCAGCTTCAGCTGCAGCTGCAGCAGCAGTCGCAGTCGCCGCACGGCCAACAGATGACGCAGCAGCAGCAGCTGGGCCAGAACGGCCTAAGGAGCACAAATAGTTTACAGTTAAGGCATAATAACGCGATGGCCGTCAGTATTGAGACCGACGTCTGACTACTAGTCAAACAAATGGAAAATGGACGAAATTTGCGCAGTGAAATGCTACGTTGGATGCCAGAAACGTCATCAAAAGCAGTCTAATTTAGAATTTTATTAATAAATACAATTAAAATATAATTATAATAATTAGTAAGCAACGTAGTTGTAAATTAAACAGCAAATGTACACAGACACAACACACACACAGACACAGTGCCAGTTCACTCAGCTTGAATTAGAGTATTTGTAGACACCAAAAAGAGTCAAATATGGACTGGCCTTCTATAGGGATTTCCTTGTTTCTCCTTTCATTTTCCTTCTGGTAATCTACACACCGAAAACACTTACACACACACGTCCACACACACTCAAAGTAAAAACTCTACTTGATACCTATGTTCAAATTTAGCAATTAACAACTAACAATCGTTAACAACAACAAAACAAAACATATAAAACCAAAAAACGAGAGAAAAAAAAAAACAAACAAAACCAAAATCTAATTATCTTAGTAGACTAATCTAATTGGAGTTTCTTCCTTTCTTTAGAAGCTAGCAAAACAAAAACAAAGAACAACAACAACCAGACAAAAACAAACATACAATATCTGCTAATTTTATTTTCATCTTTAAATTATGCTCTATTATTAAATATTAGTCAGAATATTAGTAAAACAAACGGAATTC 37

38. Drosophila Shaker K+ channel: amino acid sequence
>gi|157064|gb|AAA | potassium channel component MAAVAGLYGLGEDRQHRKKQQQQQQHQKEQLEQKEEQKKIAERKLQLREQQLQRNSLDGYGSLPKLSSQDEEGGAGHGFGGGPQHFEPIPHDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGFIKEEERPLPDNEKQRKVWLLFEYPESSQAARVVAIISVFVILLSIVIFCLETLPEFKHYKVFNTTTNGTKIEEDEVPDITDPFFLIETLCIIWFTFELTVRFLACPNKLNFCRDVMNVIDIIAIIPYFITLATVVAEEEDTLNLPKAPVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFYHREADREEMQSQNFNHVTSCSYLPGALGQHLKKSSLSESSSDIMDLDDGIDATTPGLTDHTGRHMVPFLRTQQSFEKQQLQLQLQLQQQSQSPHGQQMTQQQQLGQNGLRSTNSLQLRHNNAMAVSIETDV 38

39. Drosophila K+ channel: amino acid sequence
>gi|157064|gb|AAA | potassium channel component MAAVAGLYGLGEDRQHRKKQQQQQQHQKEQLEQKEEQKKIAERKLQLREQQLQRNSLDGYGSLPKLSSQDEEGGAGHGFGGGPQHFEPIPHDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGFIKEEERPLPDNEKQRKVWLLFEYPESSQAARVVAIISVFVILLSIVIFCLETLPEFKHYKVFNTTTNGTKIEEDEVPDITDPFFLIETLCIIWFTFELTVRFLACPNKLNFCRDVMNVIDIIAIIPYFITLATVVAEEEDTLNLPKAPVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFYHREADREEMQSQNFNHVTSCSYLPGALGQHLKKSSLSESSSDIMDLDDGIDATTPGLTDHTGRHMVPFLRTQQSFEKQQLQLQLQLQQQSQSPHGQQMTQQQQLGQNGLRSTNSLQLRHNNAMAVSIETDV
...not very illuminating at first sight, is it?
so how do we make sense of all that? 39

40. The siginficance of sequence
“Backbone”
“Side chains”
Backbone is always the same
Individual characteristics (e.g. shape) of a protein are determined by its side chains
Side chains depend on amino acid sequence 40

41. Transmembrane regions
Membrane is hydrophobic
Transmembrane domains of proteins are likely to be hydrophobic too 41

42. Drosophila K+ channel protein sequence
Predicted transmembrane domains
Hydrophobicity 42

43. Drosophila K+ channel protein
Predicted transmembrane domains
This is one of four subunits.... 43

44. Drosophila K+ channel protein
Predicted transmembrane domains
Subunit 44

45. Na+ and Ca2+ channels 45

46. Na+ and Ca2+ channels 46

47. What does it really look like?
Can be answered by X-ray crystallography
Very difficult for membrane proteins like ion channels
Finally successful: Nobel Prize 2003 (Rod MacKinnon)
Bacterial K+ channel KcsA: close relative of mammalian K+ channels
Stereo views follow... 47

48. What does it really look like?
Doyle et al 1998 48

49. What do the parts do? We can alter DNA sequences
We can express proteins and measure their properties
...So we can alter any part of an ion channel and see how its behaviour has changed
Example: the S4 region 49

50. Function of the S4 region
++++
R - arginine
K - lysine
Both are positively charged 50

51. Function of the S4 region
Something in the ion channel senses membrane voltage
Charged residues must be involved: S4 is highly charged
Is S4 the voltage sensor? How do we test?
...Change the charges: put alanine instead of lysine
If this is the voltage sensor, what will happen?
There should be a change in the amount of charge that moves when the channel opens 51

52. Charge and voltage dependence
So we can easily decide whether the charges in S4 are involved in channel opening
Here’s the result:
Charges moving when channel opens
Decrease in charge on S4 52

53. Charge and voltage dependence
Conclusion:
The charges in S4 are the ones that have to move in order to open K+ (and Na+ and Ca2+) channels
So S4 is the “voltage sensor” of the channel
Similarly we know what parts of the channel govern inactivation, ion flux, etc
...by changing parts of the protein and looking for a change in the process in question 53

54. Reading for today’s lecture:
Na+ and K+ channels
Purves et al chapter 3 (up to page 48) and chapter 4
Nicholls et al chapter 6 pages and chapters 2-3
Kandel et al chapter 6
Next lecture:
Synaptic transmission: transmitter release at the neuromuscular junction
Purves et al Chapter 5 (pages 80-95)
Nicholls et al chapter 9 (up to top of page 158 and pages )
Nicholls et al chapter 11
Kandel et al chapters 10 (page 182 onwards) and 14