30th Nov - Drug action at Ion Channels II Flashcards
Describe the nicotonic receptor superfamily
Cys-loop receptors
Assemble as a pentamer of subunits and TM2 forms the pore and is important for ion selectivity
Anion selective inhibitory receptors activated by GABA or glycine
Contain selective excitatory receptors activated by ACH of 5HT
Describe Cys loop receptors
Contain a signature extracellular sequence of 13 aa flanked by cysteines which form covalent bonds
Cys loop forms a closed loop between the ligand binding domains and channel domains
Describe nicotonic acetylcholine receptors
They are an archetypical member of the cys-loop family
2 acetylcholine molecules are required to activate it
How was the structure of nicotonic acetylcholine receptors derived?
EM images of purified ACh receptors from torpedo electric rays
How was the structure of the serotonin receptor modelled?
Using the EM model of the nicotonic ACh receptor and the crystal structure of AChBP
What is the key residue in a serotonin receptor for binding serotonin?
Y141
What is AChBP?
A protein secreted by garden pond snails which binds to ACh and has 25% sequence identity to nicotonic acetylcholine receptors
Why was it difficult to derive the structure of the serotonin receptor?
Crystallising large membrane proteins is impossible
How was the structure of the serotonin receptor derived?
Used the VHH domain of the camel heavy chain antibody to develop a nanobody which stabilises the receptor allowing crystallisation
- Made antibodies to the serotonin receptor to be used as crystallisation chaperones
- Expressed serotonin receptors in a human cell line
- Purified the serotonin receptor
- Split the receptor by trypsin proteolysis
- Crystallised the fragments with the nanobody/VHH chaperone
What is a Nanobody?
A single domain antibody which is able to selectively bind to an antigen
How was the subunit configuration of the serotonin receptor elucidated?
Used a whole cell recording to identify how different subunit compositions affected the whole cell recording.
A+B receptors were less potent than A homomers. B-E homomers do not form functional channels thus the A subunit must be present for the ion channel to function
A+C-E do not form functional channels
therefore the configuration was 2A:3B to form the channel
Are 5HT3a receptors inward rectifying?
Yes
Why does the 5HT3A and 5HT3B homomer single channel conductance vary so much and how was it investigated?
To investigate this they developed chimeric receptors in which varying regions of the homomers were swapped.
This slowly narrowed down the region responsible. Was found that there are key differences in the HA region. Part of the intracellular loop between M3 and M4, 3Arginines are conserved in A but not in A+B. Altering these arginines can alter the A homomers conductance to be the same as the AB heteromer.
As these 3 arginines create a lot of positive charge in the intracellular loop barring the way for sodium entry
Outline the expression of serotonin receptors across the body
Brain - both pre and post synaptically
GI tract - expressed in enteric NS, the vagus nerve and released from enterochromaffin cells
Peripheral sensory neurons
How does the subunit distribution of the serotonin receptor vary in the gut?
Based on the different layers:
Myenteric plexus: A, C, D, E
Submucosal plexus: A, B, D
Mucosa: A, C, D, E
