permeability changes during the action potential 1
TRANSCRIPT
Permeability changes during the action potential
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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
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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
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Hyperpolarisation: almost no current
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What ions carry the current?
Na+? K+?
•...how can we test this?•“Ion substitution”
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Na+ ions and the early transient current
i.e. current due to Na+
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Another approach to separating currents
Tetrodotoxin: toxin from Fugu puffer fish
Blocks Na+ channels
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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
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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
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Recording single ion channel currents
Patch clamp recording
•Cell attached recording
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Patch clamp recording
•Excised patch recording (“inside-out”)
Recording single ion channel currents
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Patch clamp recording:how the currents are recorded
Recording single ion channel currents
Current
Membrane patch
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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
+ + + + + +
– – – – – –
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+ + + + + +
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Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
– – – – – –
+ + + + + +
Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
– – – – – –
+ + + + + +
Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
– – – – – –
+ + + + + +
Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
– – – – – –
+ + + + + +
+ + + + + +
– – – – – –
Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
+ + + + + +
– – – – – –
Activation and inactivation of a Na+ channel
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+ + + + + +
– – – – – –
Activation and inactivation of a Na+ channel
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Ion channels are proteins
Lipid bilayer
Protein molecules
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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?
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1. What is the amino acid sequence?
•Direct protein sequencing works only for short peptides•It’s easier to determine the messenger RNA sequence
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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
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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
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Working out the mRNA sequence
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Working out the mRNA sequence
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Expressing ion channels
mRNA
Oocytes
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Working out the mRNA sequence
•But what do we screen for?•We need a starting sequence•Example:- K+ channel from Drosophila (fruit fly)
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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
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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
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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
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Drosophila Shaker K+ channel: mRNA sequence
>gi|157063|gb|M17211.1|DROCHAB D.melanogaster potassium channel (Shaker) mRNA, complete cdsGAATTCCGGAGTTTCTATCCAGACTTCAATATTTTTTTACCTCGCTCAAAACCCCCCACTCGCACTTTAAATAATAAAAAAAAGCAGGTGGTGCGTGCCGCGTAGCCGCGCGTGATTCTTGTTGTTGTTTTTTTTTTTTCGGTGAATCTCTTGTAACCATGTACCAAAGTTCTTTGCCGCGAAAACTAAAATGAAAACGAAAGTGAAAATGAGCGAATGGCAGCCGCGGCCACAGCAATCGATCCATGACACAACCAGTGACAAGCAGTCCCCCAGTGAAACCGCATCCGCATCCGAGTCCGATACCGATAAAGATTCTGAATCGGAGTGAGTGCCGCGTCCGAGAGCGTTCCCTGTCCACGTCCACCATCGGCGGAGCAGGTGTGCCTGAGGCCCACCTGGTGGCATGGCCGCCGTTGCCGGCCTCTATGGCCTTGGGGAGGATCGCCAGCACCGCAAGAAGCAGCAGCAACAGCAGCAGCACCAGAAGGAGCAGCTCGAGCAGAAGGAGGAGCAAAAGAAGATCGCCGAGCGGAAGCTGCAGCTGCGGGAGCAGCAGCTCCAGCGCAACTCCCTCGATGGTTACGGGTCTTTGCCCAAATTGAGCAGTCAAGACGAAGAAGGGGGGGCTGGTCATGGCTTTGGTGGCGGACCGCAACACTTTGAACCCATTCCTCACGATCATGATTTCTGCGAAAGAGTCGTTATAAATGTAAGCGGATTAAGGTTTGAGACACAACTACGTACGTTAAATCAATTCCCGGACACGCTGCTTGGGGATCCAGCTCGGAGATTACGGTACTTTGACCCGCTTAGAAATGAATATTTTTTTGACCGTAGTCGACCGAGCTTCGATGCGATTTTATACTATTATCAGAGTGGTGGCCGACTACGGAGACCGGTCAATGTCCCTTTAGACGTATTTAGTGAAGAAATAAAATTTTATGAATTAGGTGATCAAGCAATTAATAAATTCAGAGAGGATGAAGGCTTTATTAAAGAGGAAGAAAGACCATTACCGGATAATGAGAAACAGAGAAAAGTCTGGCTGCTCTTCGAGTATCCAGAAAGTTCGCAAGCCGCCAGAGTTGTAGCCATAATTAGTGTATTTGTTATATTGCTATCAATTGTTATATTTTGTCTAGAAACATTACCCGAATTTAAGCATTACAAGGTGTTCAATACAACAACAAATGGCACAAAAATCGAGGAAGACGAGGTGCCTGACATCACAGATCCTTTCTTCCTTATAGAAACGTTATGTATTATTTGGTTTACATTTGAACTAACTGTCAGGTTCCTCGCATGTCCGAACAAATTAAATTTCTGCAGGGATGTCATGAATGTTATCGACATAATCGCCATCATTCCGTACTTTATAACACTAGCGACTGTCGTTGCCGAAGAGGAGGATACGTTAAATCTTCCAAAAGCGCCAGTCAGTCCACAGGACAAGTCATCGAATCAGGCTATGTCCTTGGCAATATTACGAGTGATACGATTAGTTCGAGTATTTCGAATATTTAAGTTATCTAGGCATTCGAAGGGTTTACAAATATTAGGACGAACTCTGAAAGCCTCAATGCGGGAATTAGGTTTACTTATATTTTTCTTATTTATAGGCGTCGTACTCTTCTCATCGGCGGTTTATTTTGCGGAAGCTGGAAGCGAAAATTCCTTCTTCAAGTCCATACCCGATGCATTTTGGTGGGCGGTCGTTACCATGACCACCGTTGGATATGGTGACATGACGCCCGTCGGCTTCTGGGGCAAAATTGTCGGCTCTTTGTGCGTGGTCGCTGGTGTGCTGACAATCGCACTGCCGGTACCGGTTATCGTCAGTAATTTCAATTACTTCTATCACCGCGAAGCGGATCGGGAGGAGATGCAGAGCCAAAATTTCAACCACGTTACAAGTTGTTCATATTTACCTGGTGCACTAGGTCAACATTTGAAGAAATCCTCACTCTCCGAATCGTCGTCGGACATAATGGATTTGGATGATGGCATTGATGCAACCACGCCAGGTCTGACTGATCACACGGGCCGCCACATGGTGCCGTTTCTCAGGACACAGCAGTCATTCGAGAAGCAGCAGCTCCAGCTTCAGCTGCAGCTGCAGCAGCAGTCGCAGTCGCCGCACGGCCAACAGATGACGCAGCAGCAGCAGCTGGGCCAGAACGGCCTAAGGAGCACAAATAGTTTACAGTTAAGGCATAATAACGCGATGGCCGTCAGTATTGAGACCGACGTCTGACTACTAGTCAAACAAATGGAAAATGGACGAAATTTGCGCAGTGAAATGCTACGTTGGATGCCAGAAACGTCATCAAAAGCAGTCTAATTTAGAATTTTATTAATAAATACAATTAAAATATAATTATAATAATTAGTAAGCAACGTAGTTGTAAATTAAACAGCAAATGTACACAGACACAACACACACACAGACACAGTGCCAGTTCACTCAGCTTGAATTAGAGTATTTGTAGACACCAAAAAGAGTCAAATATGGACTGGCCTTCTATAGGGATTTCCTTGTTTCTCCTTTCATTTTCCTTCTGGTAATCTACACACCGAAAACACTTACACACACACGTCCACACACACTCAAAGTAAAAACTCTACTTGATACCTATGTTCAAATTTAGCAATTAACAACTAACAATCGTTAACAACAACAAAACAAAACATATAAAACCAAAAAACGAGAGAAAAAAAAAAACAAACAAAACCAAAATCTAATTATCTTAGTAGACTAATCTAATTGGAGTTTCTTCCTTTCTTTAGAAGCTAGCAAAACAAAAACAAAGAACAACAACAACCAGACAAAAACAAACATACAATATCTGCTAATTTTATTTTCATCTTTAAATTATGCTCTATTATTAAATATTAGTCAGAATATTAGTAAAACAAACGGAATTC
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Drosophila Shaker K+ channel:amino acid sequence
>gi|157064|gb|AAA28417.1| potassium channel component MAAVAGLYGLGEDRQHRKKQQQQQQHQKEQLEQKEEQKKIAERKLQLREQQLQRNSLDGYGSLPKLSSQDEEGGAGHGFGGGPQHFEPIPHDHDFCERVVINVSGLRFETQLRTLNQFPDTLLGDPARRLRYFDPLRNEYFFDRSRPSFDAILYYYQSGGRLRRPVNVPLDVFSEEIKFYELGDQAINKFREDEGFIKEEERPLPDNEKQRKVWLLFEYPESSQAARVVAIISVFVILLSIVIFCLETLPEFKHYKVFNTTTNGTKIEEDEVPDITDPFFLIETLCIIWFTFELTVRFLACPNKLNFCRDVMNVIDIIAIIPYFITLATVVAEEEDTLNLPKAPVSPQDKSSNQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAGSENSFFKSIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFYHREADREEMQSQNFNHVTSCSYLPGALGQHLKKSSLSESSSDIMDLDDGIDATTPGLTDHTGRHMVPFLRTQQSFEKQQLQLQLQLQQQSQSPHGQQMTQQQQLGQNGLRSTNSLQLRHNNAMAVSIETDV
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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?
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“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
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Transmembrane regions
Membrane is hydrophobic
Transmembrane domains of proteins are likely to be hydrophobic too
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Drosophila K+ channel protein sequence
Hyd
roph
obicity
Predicted transmembrane domains
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Drosophila K+ channel protein
Predicted transmembrane domains
This is one of four subunits....
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Drosophila K+ channel protein
Predicted transmembrane domains
Subunit
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Na+ and Ca2+ channels
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Na+ and Ca2+ channels
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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...
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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
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Function of the S4 region
R - arginineK - lysineBoth are positively charged
++++
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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
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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
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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
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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