Ion channel diversity I Flashcards
Why are ion pumps not suitable for movement of ions for generation of an AP?
Too slow and energy dependent
What are pumps used for in neurons?
Return to membrane potential after ion flux
Which conc grad direction does the generation of an AP require?
Ions to flow down electrochemical gradients
Which organism was initially studied to look for the sodium channed?
Electrophorus electricus (electric eel)
Why was the electric eel looked at for finding the Na+ channel?
Lots of electrical activity
Which organisms secrete toxins that can act at electrically gated channels?
Pufferfish and scorpion
Where do tetrodotoxin and scorpion toxin act at?
VG Na+ channels
Which organism can produce tetrodotoxin?
Pufferfish
How was the sodium channel found?
Tetrodotoxin was used as a molecular tag–> allowed purification of the channel
What was done to the Na+ channel once it was purified?
Micro Sequenced to give an AA sequence
What was done with the Na+ channel AA sequence?
Oligonucleotide probe was designed against it
What was done with the oligonucleotide probe against the Na+ channel?
Hybridized to cDNA library, some of which had the Na+ cDNA
What was learned following hybridisation of the oligonucleotide probe to the cDNA library containing Na+ cDNA?
Isolation and sequencing of the whole protein
Size of Na+ channel?
2000AAs
208kDa protein
52kDa sugar
What can xenopus oocytes be used for?
Expression of membrane protein to assess functionality
What was done with the Na+ sequence?
mRNA from the cloned channel was inserted into xenopus oocytes
What was done with the oocytes that hat Na+ channel mRNA inserted into them?
Left to incubate for several days before taking an electrical recording to see if they were sensitive to depolarization
What was observed to electrical stimulation in the xenopus oocytes that had the Na+ channel mRNA?
Depolarization, and the response was sensitive to pufferfish toxin
How was it discovered that there was smthn missing from identified mRNA and fully functional Na+ channel?
Size and kinetics of response using the cloned channel were not the same as the normal channel
What was missing from the cloned channel?
Additional subunits that are required for a fully functional channel
Why were beta subunits not identified originally?
They weren’t the associated site for tetrodotoxin
Size of beta subunits in Na+ channel?
~35kDa
What do the beta subunits contribute to in the Na+ channel?
Trafficking and folding
What is observed upon coexpression of the beta and alpha subunits?
Same results as an endogenous VG Na+ channel
Where on the VGNA+ channel are the toxin binding sites?
Alpha subunit
How does tetrodotoxin (TTX) work?
Blocks the channel from the outside
How does scorpion toxin (ScTx) work?
Alter Na+ channel properties–> slow inactivation rate
What can a hydrophobicity plot show you?
Where the hydrophobic AAs are localised–> can give info on transmembrane regions
How many transmembrane regions does the VGNa+ channel have?
24
4 repeats w/ 6 hydrophobic sections each
What is a re-entrant loop?
An area of a membranous protein that passes into the membrane but doesn’t span it entirely
