Ligand gated binding

Question 1

What is the role of inhibitory CNS channels and what are they

Answer 1

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.

Question 2

What are GluRs

Answer 2

Glutamate receptors which are cation non selective so when activate allow positive ions into the cell and depolarise the cell and are excitatory.

Question 3

What is the GlyR responsible for

Answer 3

Fast inhibitory synaptic transmission. Binding supresses the activity of the post synaptic neurons in the brainstem and spinal cord

Question 4

What is the structure of the GlyR

Answer 4

3 alpha subunits - form pore and binding site
2 beta subunits - modulate sensitivity to glycine
one pore and one binding site

Question 5

What is hyperekplexia in infants

Answer 5

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

Question 6

What is hyperekplexia in adults

Answer 6

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

Question 7

Where does the defect lie in hyperekplexia patients

Answer 7

Loss of dampening from glycine receptors - so the reflex is exaggerated - no modulation

Question 8

How is hyperekplexia treated

Answer 8

With Clonezepam which activates the GABAaR which is also a Cl channel - reduced response

Question 9

Where are the mutations found in the GlyR in hyperekplexia

Answer 9

All found in the GlyR alpha subunits, and seem to cluster in the same region of the subunits

Question 10

What is the result of the N46K mutant

Answer 10

Mutation close to the glycine binding site - around 10x more glycine required in order to produce the same current as the WT channel.

Question 11

How does the dose response curve for WT and the N46K GlyR mutant change

Answer 11

Shifts to the right - for a given concentration there is a smaller response.

Question 12

What is affinity

Answer 12

The tendency of the ligand to bind its receptor

Question 13

What is efficacy

Answer 13

The tendency of a ligand to activate its receptor once bound

Question 14

How does a change in affinity present itself on a dose response curve (full agonist)

Answer 14

Sideways shift

Question 15

How does a change of efficacy present itself on a dose response curve (full agonist)

Answer 15

Sideways shift - with a partial agonist you see a sideways shift AND a lower maximal response

Question 16

What was the partial agonist for the glycine receptor

Answer 16

Taurine

Question 17

What was the effect of using taurine on the dose response curve

Answer 17

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

Question 18

What is the difference between deactivation and desensitisation of ligand binding channels

Answer 18

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.

Question 19

What is the benefit of looking at current sizes for 200ms compared to 2ms in GlyRs

Answer 19

200ms allows for study of deactivation with desensitisation

2ms only shows deactivation

Question 20

What was the result of glycine exposure for 200ms of glycine on WT and mutant channels

Answer 20

time to activation very similar - faster decrease in current during deactivation + desensitisation

Question 21

What was the result of glycine exposure for 2ms of glycine on WT and N46K mutant

Answer 21

Normal activation but extremely quick deactivation (90% current down to 10%)

Question 22

What is the mouse model of hyperekplexia

Answer 22

Shaky Q177K

with hind feet and limb clenching as well as motor defects beyond 2 weeks of age.

Question 23

How does the righting time in shaky mice change after 2 weeks

Answer 23

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

Question 24

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

Answer 24

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

Question 25

What is diazepam

Answer 25

GABAaR agonist - Cl channel

Question 26

What is gephyrin - how was it used

Answer 26

Post synaptic membrane marker - Co-expression study of gephrin with GlyR - would expect considerable overlap

Question 27

How does shaky mice GlyR expression change

Answer 27

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.

Question 28

What changes are seen in the dose response curve in shaky mice to glycine treatment

Answer 28

Shift to the right

Question 29

How does deactivation/desensitisation change in the shaky mutant

Answer 29

Activation is the same but deactivation/desensitisation is faster

Question 30

How do brainstem recordings of the mice change between WT and shaky mice

Answer 30

Add glycine and record chloride currents in the excitable neurons - very little in shaky mice seen