Ion channel diversity Flashcards
What are channelopathies?
Mutations in Na+ or K+ channels can produce hyperexcitable neurones - resulting in epilepsy.
Principal target of phenytoin (anti-epileptic drug) is inhibition of VA Na+ channels
Retigabine is anti-epileptic drug with principal target of VA K+ channels
Why are ion channels and not pumps used for APs?
Channels can move ions considerably faster….
= 10^7 - 10^8 ions per second.
= Pumps = 10^5 ions per second.
Channel needs to open and close within 2 mili seconds.
= speed necessary for an AP can only be provided by ion channel flux, not pumps.
What are the requirements of ion channels in AP?
Rapid inactivation mechanism - Channel needs to open + close within 1-2 miliseconds.
Ion selectivity.
Voltage dependent activation and inactivation.
= Produce rapid and reporudicble patterns of selective ion fluxes..
What are some examples of potent VA NA+ channel blockers….
What animal is a rich source of VA Na+ channels?
Tetrodo toxin = Puffer Fish = blocks channel from OUTSIDE
Scorpion Toxin… = slows inactivation rate.
Electric Eel!
How was the Sodium channel cloned?
Affinity purification:
Tetrodotoxin was used as a tag for sodium channels in electric eel tissue as it selectively inhibits/tags the channel.
Tagged channels were then selectively purified.
A small region of the channel’s AA sequence was sequenced and an oligonucleotide probe was designed, complementary to the AA sequence.
Oligonucleotide probe was hybridised to cDNA library, containing Na+ channel cDNA.
The entire sodium channel sequence was isolated and sequenced.
How were cloned Na+ channels expressed + Why in this model?
mRNA of cloned Na+ channel cDNA was injected into Xenopus Laevis oocytes.
= As oocytes, Xenopus laevis do not express ANY cell surface receptors…
Applying depolarisation resulted in response that confirmed Na+ channel was Voltage-dependent.
But could be blocked by tetrodotoxin.
What was discovered about the kinetics of the cloned Na+ channel
Applying depolarisation resulted in response that confirmed Na+ channel was Voltage-dependent.
But could be blocked by tetrodotoxin.
When only cloned human Na+ channel RNA was injected - size and kinetics of inward rising current were not the same as with TOTAL RNA injection (including auxilary units)…
Fully functional Na+ channels require auxillary beta subunits.
Purified channels from rat brain reveealed a large subunit, but also two much smaller auxilary Beta subunits.
Co-expression with beta subunits increased activity of channel!
What were the original models of Na+ channel made based on original biochemical data?
Larger alpha subunit was responsible for channel voltage-dependency, whilst smaller beta subunits modulate the kinetics and voltage dependence of channel activation/inactivation aswell as functional folding of alpha subunit for TOTAL activity…
How are the TM segments arranged in the alpha subunit of Na+ channel?
Hydrophobicity plot predicts 4 repeats of 6 hydrophobic sections long enough to span PM.
= 24 TM domains!
Intracellular N terminus - intracellular loops and finishes with intracellular C terminus.
S1-4 are voltage-sensitive, especially S4.
S5 and S6 contain pore elements required for ion selectivity.
Inactivation gate.
Long intracellular loops are areas of regulation - controlled mainly by phosphorylation, as well as drug binding sites.
Entirely seprarate auxilliary beta subunits - coded by independent genes.
How are ion channels conserved evolutionary?
Many K+ channels, fewer Na+ and Ca2+ channels.
10 VG Na+ and 10 VG Ca2+ channels.
Bacterial Na+ channel believed to be common ancestor to eukaryotic Na+ and Ca2+ channels.
Homologous but contains fewer intracellular regulatory loops than eukaryotes.
What are some important structural details regarding selectivity filter and voltage sensors in Na+ channel?
Within the central pore, there is first an extracellular funnel, followed by a selectivity filter to determine which cation can pass through.
= P loop creates selectivity filter giving specificity to only Na+
After the selectivity filter, there is a central cavitiy filled with water molecules, then the activation gate.
Voltage sensor, particularly S4, contain Arginines - R1 - R4 = voltage sensing arginines.
AT physiological pH, these residues are positively charged.
When membrane depolarises to threshold, arginine residues R1 to R4 are moved outwardly and pull on the S4-S5 linker to open the channel
= Voltage sensing arginines R1-R4 are moved outwardly and pull on S4-S5 linker to open the channel in response to change in electric field
What is the sliding helix/helical twist model?
Common to all voltage-dependent ion channels like VA Na+, Ca2+, K+.
Electro-mechanical coupling opens voltage-activated ion channel via Sliding helix/Helical twist model:
In resting state, negatively charged ions in INC (intracellulra negative clusters) associated with positively charged arginines in S4 domain (residues R1-R4).
Depolarisation causes of movement of + charge to outside and creates a small “gating current” preceding Na+ entry!!.
Hydrophobic seal keeps channel closed on resting state….
How are Na+ channels inactivated?
Hinged Lid Mechanism.
an intracellular 4 amino acid long motif = IFMT in the cytoplasmic loop/ S3/S4 linker occludes the ion pore through a hinged lid mechanism.
IFMT acts as hydrophobic latch…
What is the unique signature sequence to all voltage-activated K+ channels?
Five AA signature sequence
GYGVT.
How does the Na+ channel compared to K+ channels?
4 independent subunits assemble to make VA K+ channel.
Na+ channels alpha domain composed of 1 large polypeptide of 4 repeated TM domains.
Similarly, voltage sensing area in 4th domain of K+ channel, with +vely charged amino acids too!.
Different selectivity filter
Inactivation via Ball and Chain mechanism - N terminus of each subunit of channel tetramer to inactivate channel = K+
Inactivation via Hinged Lid mechanism - IFMT in cytoplasmic S3-S4 linker occludes ion pore acting as hydrophobic latch.
