Sodium channels Flashcards
what are the key properties that differentiate pumps vs channels?
pumps:
- energy dependent
- ions transported against conc grad
- slow <10^5 ions/sec
channels:
- ions flow down electrochemical gradient
- fast 10^7-10^8 ions/sec
what are the ion channel properties required for an AP?
voltage dependence
ion selectivity
inactivation mechanism of open channel
which 2 animals were using in thepurifying and cloning sodium channels?
electric eels
- rich source of Na channels
Puffer fish
- produce Tetrodotoxin
= potent inhibitor to be used as a tag
describe the process of Na+ channel cloning
- remove sodium channel protein from electric eel
- purify protein using tetrodotoxin as a tag
- micro sequence the pure protein
- > amino acid sequence of small region of channel protein - use to design an oligonucleotide probe corresponding to amino acid sequence
- hybridise to cDNA library containing Na+ channel DNA
- isolate + sequence Na+ channel cDNA
describe how expression of Na+ channel protein in cloned cDNA is confirmed
what else did this confirm?
- mRNA from cloned Na+ channel cDNA injected into Xenopus oocytes
- incubate for several days
- channels expressed and inserted into oocyte membrane respond
- current recorded from functional channels
if when depolarisation is applied and an appropriate response is recorded
= confirms cloned cDNA encodes a voltage-dependent Na+ channel
the response could be blocked by tetrodotoxin
how were sodium channels identified in purified rat brain?
SDS PAGE
- confirmed 1 large alpha subunit
- 2 smaller beta subunits
Photo-affinity labelling
- Scorpion toxin (ScTx) used to label the channel
- > labels band the same size as alpha band from SDS PAGE
describe the structure of the rat brain sodium channel
how does TTX and ScTx affect the channel?
large alpha subunit with 2 beta subunits attached
- b subunits speed up activation
intracellular phosphorylation sites attached to the alpha subunit
TTX blocks channel from outside
ScTx alters the channel’s properties
= slows inactivation rate
what is a hydrophobicity index?
in a protein, where are hydrophobic and hydrophilic amino acids found?
a measure fo the relative hydrophobicity of an amino acid
or how water-soluble an amino acid is
hydrophobic = interior hydrophilic = in contact with aqueous environment
what did the hydrophobicity plot for the alpha unit reveal?
4 domains of 6 hydrophobic transmembrane spans
there is a re-entrant loop between spans 5 and 6
the pore is between spans 5 and 6
the voltage sensing domain is in transmembrane segment 4
intracellular loops between repeats 1+2, and 2+3
contain P sites that can modulate activity
intracellular loop between 3+4
important for inactivation
what are the features of the beta subunits?
single transmembrane spans
modulate the kinetics and voltage dependence of sodium channel activation/inactivation
what did the crystal structure of the Na channel reveal?
the P loop creates a ‘selectivity filter’ that gives specificity for Na+
describe the voltage-sensor S4 sequence
positively charged amino acids (Arg, Lys) are conserved in S4 of different channel proteins
these line up on the outside of the S4 alpha helix
how does S4 respond to depolarisation?
as neurone is depolarised
-> inside is more + and voltage sensor moves to create gating current
the movement causes conformational change + creates a small ‘gating current’ that allows Na+ entry (= channel opening)
what does Na+ channel inactivation result in?
what does this limit?
during a long period of depolarisation, why is this essential?
closes the channel + prevents it reopening
= allows time for recovery of ion gradients + return to resting membrane potential
frequency of action potential firing
protects neurone from a breakdown of ionic gradients that can lead to cell death
describe the hinged lid mechanism for Na+ channel inactivation
mediated by a 4-amino acid motif IFMT, in the domain 3/4 linker
this linker can block the pore, with the IFMT motif serving as a hydrophobic ‘latch’ by binding to the channel
