Ligand gated binding Flashcards
What is the role of inhibitory CNS channels and what are they
GlyR and GABAaR are both chloride channels, when activated they allow Cl to move into the cell, bringing the Vm down towards ECl which is negative.
What are GluRs
Glutamate receptors which are cation non selective so when activate allow positive ions into the cell and depolarise the cell and are excitatory.
What is the GlyR responsible for
Fast inhibitory synaptic transmission. Binding supresses the activity of the post synaptic neurons in the brainstem and spinal cord
What is the structure of the GlyR
3 alpha subunits - form pore and binding site
2 beta subunits - modulate sensitivity to glycine
one pore and one binding site
What is hyperekplexia in infants
Autosomal dominant and recessive forms - hypertonia - SIDS
Enhanced startle reflex
Auditory and tactile stimuli causes this response
demonstarte apnoea - muscles in the throat relax and stop breathing for a period of time before starting again
life threatening
What is hyperekplexia in adults
Hypertonia disappears
Enhanced startle reflex remains for auditory and tactile stimuli
Falls and injuries are likely as a consequence but not considered to be life threatening
Where does the defect lie in hyperekplexia patients
Loss of dampening from glycine receptors - so the reflex is exaggerated - no modulation
How is hyperekplexia treated
With Clonezepam which activates the GABAaR which is also a Cl channel - reduced response
Where are the mutations found in the GlyR in hyperekplexia
All found in the GlyR alpha subunits, and seem to cluster in the same region of the subunits
What is the result of the N46K mutant
Mutation close to the glycine binding site - around 10x more glycine required in order to produce the same current as the WT channel.
How does the dose response curve for WT and the N46K GlyR mutant change
Shifts to the right - for a given concentration there is a smaller response.
What is affinity
The tendency of the ligand to bind its receptor
What is efficacy
The tendency of a ligand to activate its receptor once bound
How does a change in affinity present itself on a dose response curve (full agonist)
Sideways shift
How does a change of efficacy present itself on a dose response curve (full agonist)
Sideways shift - with a partial agonist you see a sideways shift AND a lower maximal response
What was the partial agonist for the glycine receptor
Taurine
What was the effect of using taurine on the dose response curve
shift in dose response but no change in maximum current so the change due to the mutation is likely due to a change in affinity of glycine for GlyR
What is the difference between deactivation and desensitisation of ligand binding channels
Key feature of ligand ion channels - expose channel the channel will activate and then completely desensitise, wash off ligand and reintroduce and nothing will happen for a given time until desensitisation has been overcome.
What is the benefit of looking at current sizes for 200ms compared to 2ms in GlyRs
200ms allows for study of deactivation with desensitisation
2ms only shows deactivation
What was the result of glycine exposure for 200ms of glycine on WT and mutant channels
time to activation very similar - faster decrease in current during deactivation + desensitisation
What was the result of glycine exposure for 2ms of glycine on WT and N46K mutant
Normal activation but extremely quick deactivation (90% current down to 10%)
What is the mouse model of hyperekplexia
Shaky Q177K
with hind feet and limb clenching as well as motor defects beyond 2 weeks of age.
How does the righting time in shaky mice change after 2 weeks
If knocked over, young animals take a similar amount of time to stand as WT mice. After two weeks the mice struggle to stand back up
What difference is seen in WT compared to shaky mice when placed on a rod and how can the time of the shaky mice be improved
Shaky mice unable to stay on the rod compared to WT - treatment with diazepam increases the time they are able to stay on the rod but not to WT levels
What is diazepam
GABAaR agonist - Cl channel
What is gephyrin - how was it used
Post synaptic membrane marker - Co-expression study of gephrin with GlyR - would expect considerable overlap
How does shaky mice GlyR expression change
More total protein in the mutant cells but less overlap so less GlyR at the membrane, synaptic location disrupted - suggests the mutation disrupts the trafficking of the channel to the post synaptic membrane.
What changes are seen in the dose response curve in shaky mice to glycine treatment
Shift to the right
How does deactivation/desensitisation change in the shaky mutant
Activation is the same but deactivation/desensitisation is faster
How do brainstem recordings of the mice change between WT and shaky mice
Add glycine and record chloride currents in the excitable neurons - very little in shaky mice seen
