Permeability changes during the action potential

1

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

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

So what do we see on depolarisation?

Early transient inward current

Late sustained outward current

4

Hyperpolarisation: almost no current

5

What ions carry the current?

Na+? K+?

•...how can we test this?•“Ion substitution”

6

Na+ ions and the early transient current

i.e. current due to Na+

7

Another approach to separating currents

Tetrodotoxin: toxin from Fugu puffer fish

Blocks Na+ channels

8

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

Characteristics of Na+ and K+ currents

Na+ current:•Very fast activation•Fast inactivation

K+ current:•Slower activation than Na+ current•No inactivation on timescale of an AP

10

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

Recording single ion channel currents

Patch clamp recording

•Cell attached recording

12

Patch clamp recording

•Excised patch recording (“inside-out”)

Recording single ion channel currents

13

Patch clamp recording:how the currents are recorded

Recording single ion channel currents

Current

Membrane patch

14

Na+ channel activation and inactivation

closed

open

–90 mV

-60 mVVoltage (Em)

Current

Depolarisation opens the channel:activation

It closes again spontaneously:inactivation

Reid et al 1991 15

Activation and inactivation of a Na+ channel

+ + + + + +

– – – – – –

16

+ + + + + +

– – – – – –

– – – – – –

+ + + + + +

Activation and inactivation of a Na+ channel

17

+ + + + + +

– – – – – –

– – – – – –

+ + + + + +

Activation and inactivation of a Na+ channel

18

+ + + + + +

– – – – – –

– – – – – –

+ + + + + +

Activation and inactivation of a Na+ channel

19

+ + + + + +

– – – – – –

– – – – – –

+ + + + + +

Activation and inactivation of a Na+ channel

20

+ + + + + +

– – – – – –

– – – – – –

+ + + + + +

+ + + + + +

– – – – – –

Activation and inactivation of a Na+ channel

21

+ + + + + +

– – – – – –

+ + + + + +

– – – – – –

Activation and inactivation of a Na+ channel

22

+ + + + + +

– – – – – –

Activation and inactivation of a Na+ channel

23

Ion channels are proteins

Lipid bilayer

Protein molecules

24

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

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

mRNA and protein sequence

•Three bases in mRNA code for one amino acid in the protein

•So:•If we know the mRNA sequence we can work out the amino acid sequence

27

Working out the mRNA sequence

•Beads with TTT attached: mRNA sticks•Wash off all the rest•Then separate mRNA from beads 28

Working out the mRNA sequence

29

Working out the mRNA sequence

30

Working out the mRNA sequence

31

Expressing ion channels

mRNA

Oocytes

32

Working out the mRNA sequence

•But what do we screen for?•We need a starting sequence•Example:- K+ channel from Drosophila (fruit fly)

33

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

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

Drosophila K+ channel

Region 16F on X chromosome

Starting clone

Overlapping clone 1

Probe with end onlyOverlapping clone 2

Probe with end onlyOverlapping 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

Drosophila Shaker K+ channel: mRNA sequence

>gi|157063|gb|M17211.1|DROCHAB D.melanogaster potassium channel (Shaker) mRNA, complete cdsGAATTCCGGAGTTTCTATCCAGACTTCAATATTTTTTTACCTCGCTCAAAACCCCCCACTCGCACTTTAAATAATAAAAAAAAGCAGGTGGTGCGTGCCGCGTAGCCGCGCGTGATTCTTGTTGTTGTTTTTTTTTTTTCGGTGAATCTCTTGTAACCATGTACCAAAGTTCTTTGCCGCGAAAACTAAAATGAAAACGAAAGTGAAAATGAGCGAATGGCAGCCGCGGCCACAGCAATCGATCCATGACACAACCAGTGACAAGCAGTCCCCCAGTGAAACCGCATCCGCATCCGAGTCCGATACCGATAAAGATTCTGAATCGGAGTGAGTGCCGCGTCCGAGAGCGTTCCCTGTCCACGTCCACCATCGGCGGAGCAGGTGTGCCTGAGGCCCACCTGGTGGCATGGCCGCCGTTGCCGGCCTCTATGGCCTTGGGGAGGATCGCCAGCACCGCAAGAAGCAGCAGCAACAGCAGCAGCACCAGAAGGAGCAGCTCGAGCAGAAGGAGGAGCAAAAGAAGATCGCCGAGCGGAAGCTGCAGCTGCGGGAGCAGCAGCTCCAGCGCAACTCCCTCGATGGTTACGGGTCTTTGCCCAAATTGAGCAGTCAAGACGAAGAAGGGGGGGCTGGTCATGGCTTTGGTGGCGGACCGCAACACTTTGAACCCATTCCTCACGATCATGATTTCTGCGAAAGAGTCGTTATAAATGTAAGCGGATTAAGGTTTGAGACACAACTACGTACGTTAAATCAATTCCCGGACACGCTGCTTGGGGATCCAGCTCGGAGATTACGGTACTTTGACCCGCTTAGAAATGAATATTTTTTTGACCGTAGTCGACCGAGCTTCGATGCGATTTTATACTATTATCAGAGTGGTGGCCGACTACGGAGACCGGTCAATGTCCCTTTAGACGTATTTAGTGAAGAAATAAAATTTTATGAATTAGGTGATCAAGCAATTAATAAATTCAGAGAGGATGAAGGCTTTATTAAAGAGGAAGAAAGACCATTACCGGATAATGAGAAACAGAGAAAAGTCTGGCTGCTCTTCGAGTATCCAGAAAGTTCGCAAGCCGCCAGAGTTGTAGCCATAATTAGTGTATTTGTTATATTGCTATCAATTGTTATATTTTGTCTAGAAACATTACCCGAATTTAAGCATTACAAGGTGTTCAATACAACAACAAATGGCACAAAAATCGAGGAAGACGAGGTGCCTGACATCACAGATCCTTTCTTCCTTATAGAAACGTTATGTATTATTTGGTTTACATTTGAACTAACTGTCAGGTTCCTCGCATGTCCGAACAAATTAAATTTCTGCAGGGATGTCATGAATGTTATCGACATAATCGCCATCATTCCGTACTTTATAACACTAGCGACTGTCGTTGCCGAAGAGGAGGATACGTTAAATCTTCCAAAAGCGCCAGTCAGTCCACAGGACAAGTCATCGAATCAGGCTATGTCCTTGGCAATATTACGAGTGATACGATTAGTTCGAGTATTTCGAATATTTAAGTTATCTAGGCATTCGAAGGGTTTACAAATATTAGGACGAACTCTGAAAGCCTCAATGCGGGAATTAGGTTTACTTATATTTTTCTTATTTATAGGCGTCGTACTCTTCTCATCGGCGGTTTATTTTGCGGAAGCTGGAAGCGAAAATTCCTTCTTCAAGTCCATACCCGATGCATTTTGGTGGGCGGTCGTTACCATGACCACCGTTGGATATGGTGACATGACGCCCGTCGGCTTCTGGGGCAAAATTGTCGGCTCTTTGTGCGTGGTCGCTGGTGTGCTGACAATCGCACTGCCGGTACCGGTTATCGTCAGTAATTTCAATTACTTCTATCACCGCGAAGCGGATCGGGAGGAGATGCAGAGCCAAAATTTCAACCACGTTACAAGTTGTTCATATTTACCTGGTGCACTAGGTCAACATTTGAAGAAATCCTCACTCTCCGAATCGTCGTCGGACATAATGGATTTGGATGATGGCATTGATGCAACCACGCCAGGTCTGACTGATCACACGGGCCGCCACATGGTGCCGTTTCTCAGGACACAGCAGTCATTCGAGAAGCAGCAGCTCCAGCTTCAGCTGCAGCTGCAGCAGCAGTCGCAGTCGCCGCACGGCCAACAGATGACGCAGCAGCAGCAGCTGGGCCAGAACGGCCTAAGGAGCACAAATAGTTTACAGTTAAGGCATAATAACGCGATGGCCGTCAGTATTGAGACCGACGTCTGACTACTAGTCAAACAAATGGAAAATGGACGAAATTTGCGCAGTGAAATGCTACGTTGGATGCCAGAAACGTCATCAAAAGCAGTCTAATTTAGAATTTTATTAATAAATACAATTAAAATATAATTATAATAATTAGTAAGCAACGTAGTTGTAAATTAAACAGCAAATGTACACAGACACAACACACACACAGACACAGTGCCAGTTCACTCAGCTTGAATTAGAGTATTTGTAGACACCAAAAAGAGTCAAATATGGACTGGCCTTCTATAGGGATTTCCTTGTTTCTCCTTTCATTTTCCTTCTGGTAATCTACACACCGAAAACACTTACACACACACGTCCACACACACTCAAAGTAAAAACTCTACTTGATACCTATGTTCAAATTTAGCAATTAACAACTAACAATCGTTAACAACAACAAAACAAAACATATAAAACCAAAAAACGAGAGAAAAAAAAAAACAAACAAAACCAAAATCTAATTATCTTAGTAGACTAATCTAATTGGAGTTTCTTCCTTTCTTTAGAAGCTAGCAAAACAAAAACAAAGAACAACAACAACCAGACAAAAACAAACATACAATATCTGCTAATTTTATTTTCATCTTTAAATTATGCTCTATTATTAAATATTAGTCAGAATATTAGTAAAACAAACGGAATTC

37

Drosophila Shaker K+ channel:amino acid sequence

>gi|157064|gb|AAA28417.1| potassium channel component MAAVAGLYGLGEDRQHRKKQQQQQQHQKEQLEQKEEQKKIAERKLQLREQQLQRNSLDGYGSLPKLSSQDEEGGAGHGFGGGPQHFEPIPHDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGFIKEEERPLPDNEKQRKVWLLFEYPESSQAARVVAIISVFVILLSIVIFCLETLPEFKHYKVFNTTTNGTKIEEDEVPDITDPFFLIETLCIIWFTFELTVRFLACPNKLNFCRDVMNVIDIIAIIPYFITLATVVAEEEDTLNLPKAPVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFYHREADREEMQSQNFNHVTSCSYLPGALGQHLKKSSLSESSSDIMDLDDGIDATTPGLTDHTGRHMVPFLRTQQSFEKQQLQLQLQLQQQSQSPHGQQMTQQQQLGQNGLRSTNSLQLRHNNAMAVSIETDV

38

Drosophila K+ channel: amino acid sequence

>gi|157064|gb|AAA28417.1| potassium channel component MAAVAGLYGLGEDRQHRKKQQQQQQHQKEQLEQKEEQKKIAERKLQLREQQLQRNSLDGYGSLPKLSSQDEEGGAGHGFGGGPQHFEPIPHDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGFIKEEERPLPDNEKQRKVWLLFEYPESSQAARVVAIISVFVILLSIVIFCLETLPEFKHYKVFNTTTNGTKIEEDEVPDITDPFFLIETLCIIWFTFELTVRFLACPNKLNFCRDVMNVIDIIAIIPYFITLATVVAEEEDTLNLPKAPVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFYHREADREEMQSQNFNHVTSCSYLPGALGQHLKKSSLSESSSDIMDLDDGIDATTPGLTDHTGRHMVPFLRTQQSFEKQQLQLQLQLQQQSQSPHGQQMTQQQQLGQNGLRSTNSLQLRHNNAMAVSIETDV

•...not very illuminating at first sight, is it?•so how do we make sense of all that?

39

“Backbone” “Side chains”

The siginficance of sequence

•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

Transmembrane regions

Membrane is hydrophobic

Transmembrane domains of proteins are likely to be hydrophobic too

41

Drosophila K+ channel protein sequence

Hyd

roph

obicity

Predicted transmembrane domains

42

Drosophila K+ channel protein

Predicted transmembrane domains

This is one of four subunits....

43

Drosophila K+ channel protein

Predicted transmembrane domains

Subunit

44

Na+ and Ca2+ channels

45

Na+ and Ca2+ channels

46

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

What does it really look like?

Doyle et al 1998 48

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

Function of the S4 region

R - arginineK - lysineBoth are positively charged

++++

50

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

Charge and voltage dependence

•So we can easily decide whether the charges in S4 are involved in channel opening•Here’s the result: C

harg

es mo

ving

wh

en ch

ann

el op

ens

Decrease in charge on S4

52

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

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 94-102 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 160-162)

•Nicholls et al chapter 11•Kandel et al chapters 10 (page 182 onwards) and 14