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? Late sustained outward current Early transient inward current 4

Hyperpolarisation: almost no current 5

What ions carry the current? K+? Na+? ...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 Slower activation than Na+ current No inactivation on timescale of an AP Na+ current: Very fast activation Fast inactivation 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

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

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

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

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 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

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 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

Drosophila Shaker K+ channel: mRNA sequence >gi|157063|gb|M17211.1|DROCHAB D.melanogaster potassium channel (Shaker) mRNA, complete cds GAATTCCGGAGTTTCTATCCAGACTTCAATATTTTTTTACCTCGCTCAAAACCCCCCACTCGCACTTTAAATAATAAAAAAAAGCAGGTGGTGCGTGCCGCGTAGCCGCGCGTGATTCTTGTTGTTGTTTTTTTTTTTTCGGTGAATCTCTTGTAACCATGTACCAAAGTTCTTTGCCGCGAAAACTAAAATGAAAACGAAAGTGAAAATGAGCGAATGGCAGCCGCGGCCACAGCAATCGATCCATGACACAACCAGTGACAAGCAGTCCCCCAGTGAAACCGCATCCGCATCCGAGTCCGATACCGATAAAGATTCTGAATCGGAGTGAGTGCCGCGTCCGAGAGCGTTCCCTGTCCACGTCCACCATCGGCGGAGCAGGTGTGCCTGAGGCCCACCTGGTGGCATGGCCGCCGTTGCCGGCCTCTATGGCCTTGGGGAGGATCGCCAGCACCGCAAGAAGCAGCAGCAACAGCAGCAGCACCAGAAGGAGCAGCTCGAGCAGAAGGAGGAGCAAAAGAAGATCGCCGAGCGGAAGCTGCAGCTGCGGGAGCAGCAGCTCCAGCGCAACTCCCTCGATGGTTACGGGTCTTTGCCCAAATTGAGCAGTCAAGACGAAGAAGGGGGGGCTGGTCATGGCTTTGGTGGCGGACCGCAACACTTTGAACCCATTCCTCACGATCATGATTTCTGCGAAAGAGTCGTTATAAATGTAAGCGGATTAAGGTTTGAGACACAACTACGTACGTTAAATCAATTCCCGGACACGCTGCTTGGGGATCCAGCTCGGAGATTACGGTACTTTGACCCGCTTAGAAATGAATATTTTTTTGACCGTAGTCGACCGAGCTTCGATGCGATTTTATACTATTATCAGAGTGGTGGCCGACTACGGAGACCGGTCAATGTCCCTTTAGACGTATTTAGTGAAGAAATAAAATTTTATGAATTAGGTGATCAAGCAATTAATAAATTCAGAGAGGATGAAGGCTTTATTAAAGAGGAAGAAAGACCATTACCGGATAATGAGAAACAGAGAAAAGTCTGGCTGCTCTTCGAGTATCCAGAAAGTTCGCAAGCCGCCAGAGTTGTAGCCATAATTAGTGTATTTGTTATATTGCTATCAATTGTTATATTTTGTCTAGAAACATTACCCGAATTTAAGCATTACAAGGTGTTCAATACAACAACAAATGGCACAAAAATCGAGGAAGACGAGGTGCCTGACATCACAGATCCTTTCTTCCTTATAGAAACGTTATGTATTATTTGGTTTACATTTGAACTAACTGTCAGGTTCCTCGCATGTCCGAACAAATTAAATTTCTGCAGGGATGTCATGAATGTTATCGACATAATCGCCATCATTCCGTACTTTATAACACTAGCGACTGTCGTTGCCGAAGAGGAGGATACGTTAAATCTTCCAAAAGCGCCAGTCAGTCCACAGGACAAGTCATCGAATCAGGCTATGTCCTTGGCAATATTACGAGTGATACGATTAGTTCGAGTATTTCGAATATTTAAGTTATCTAGGCATTCGAAGGGTTTACAAATATTAGGACGAACTCTGAAAGCCTCAATGCGGGAATTAGGTTTACTTATATTTTTCTTATTTATAGGCGTCGTACTCTTCTCATCGGCGGTTTATTTTGCGGAAGCTGGAAGCGAAAATTCCTTCTTCAAGTCCATACCCGATGCATTTTGGTGGGCGGTCGTTACCATGACCACCGTTGGATATGGTGACATGACGCCCGTCGGCTTCTGGGGCAAAATTGTCGGCTCTTTGTGCGTGGTCGCTGGTGTGCTGACAATCGCACTGCCGGTACCGGTTATCGTCAGTAATTTCAATTACTTCTATCACCGCGAAGCGGATCGGGAGGAGATGCAGAGCCAAAATTTCAACCACGTTACAAGTTGTTCATATTTACCTGGTGCACTAGGTCAACATTTGAAGAAATCCTCACTCTCCGAATCGTCGTCGGACATAATGGATTTGGATGATGGCATTGATGCAACCACGCCAGGTCTGACTGATCACACGGGCCGCCACATGGTGCCGTTTCTCAGGACACAGCAGTCATTCGAGAAGCAGCAGCTCCAGCTTCAGCTGCAGCTGCAGCAGCAGTCGCAGTCGCCGCACGGCCAACAGATGACGCAGCAGCAGCAGCTGGGCCAGAACGGCCTAAGGAGCACAAATAGTTTACAGTTAAGGCATAATAACGCGATGGCCGTCAGTATTGAGACCGACGTCTGACTACTAGTCAAACAAATGGAAAATGGACGAAATTTGCGCAGTGAAATGCTACGTTGGATGCCAGAAACGTCATCAAAAGCAGTCTAATTTAGAATTTTATTAATAAATACAATTAAAATATAATTATAATAATTAGTAAGCAACGTAGTTGTAAATTAAACAGCAAATGTACACAGACACAACACACACACAGACACAGTGCCAGTTCACTCAGCTTGAATTAGAGTATTTGTAGACACCAAAAAGAGTCAAATATGGACTGGCCTTCTATAGGGATTTCCTTGTTTCTCCTTTCATTTTCCTTCTGGTAATCTACACACCGAAAACACTTACACACACACGTCCACACACACTCAAAGTAAAAACTCTACTTGATACCTATGTTCAAATTTAGCAATTAACAACTAACAATCGTTAACAACAACAAAACAAAACATATAAAACCAAAAAACGAGAGAAAAAAAAAAACAAACAAAACCAAAATCTAATTATCTTAGTAGACTAATCTAATTGGAGTTTCTTCCTTTCTTTAGAAGCTAGCAAAACAAAAACAAAGAACAACAACAACCAGACAAAAACAAACATACAATATCTGCTAATTTTATTTTCATCTTTAAATTATGCTCTATTATTAAATATTAGTCAGAATATTAGTAAAACAAACGGAATTC 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

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

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

Drosophila K+ channel protein sequence Predicted transmembrane domains Hydrophobicity 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 - arginine K - lysine Both 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: Charges moving when channel opens 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