Ligand-gated ion channels 2 Flashcards
1
Q
What are 5 drug targets of GABAARs
A
1.Benzodiazepines
2. Alcohol
3. Barbiturates
4. Anaesthetics
5. Neurosteroids
2
Q
What action do BZs have
A
- Multiple actions e.g. anxiety relieving, anticonvulsants
3
Q
What action does alcohol have
A
- Ethanol binds to GABAARs (as well as ACh, 5HT, NMDA-Rs)
- NOT selective for GABAaRS
4
Q
What action do barbiturates have
A
- pentobarbital has sedative/anticonvulsant effects
5
Q
What action does anaesthetics have
A
- e.g. etomidate & propofol are volatile anaesthetics that act at GABAARs
6
Q
What are examples of neurosteroids
A
- Metabolites of progesterone & deoxycorticosterone
7
Q
What are positive modulators
A
- enhance agonist induced action
- Some BZs increase GABA induced Cl- channel opening by increasing efficacy of GABA for channel opening by increasing open frequency probability
8
Q
What are negative modulators
A
- reduce agonist-induced action
- e.g. Some BZs decrease GABA induced Cl- channel opening
9
Q
What do barbiturates do and compare to BZs
A
- barbiturates (e.g. pentobarbital) bind to a different site and prolong lifetime of open state – even in the absence of GABA
- BZs are allosteric modulators – therapeutic advance on barbiturates as safer
10
Q
Why are allosteric modulators used as LGIC drug targets rather than antagonists?
A
- Large repertoire of allosteric modulators for a particular receptor
- Binding of an allosteric modulator is not restricted to the ligand-binding site or the ion pore
- Orthosteric antagonists can cause insurmountable blockade of the receptor and obliterate the physiological response
- Positive modulators could “turn up” an inhibitory pathway
- Negative allosteric modulators reduce agonist sensitivity and activity
- Can target particular receptor subtypes depending on subunit composition
- Keep the temporal and spatial activation of the receptors- only activates when GABAa is present
- Understanding of subunits gives potential for increased selectivity
11
Q
What is the allosteric site
A
- Benzodiazepines are allosteric modulators that bind at the interface between the a/g subunit – allosteric site
12
Q
Describe structure of BZs
A
- The core of all BZ ligands is a benzene ring joined to a 7-membered 1,4-diazepine ring
- R side groups influence affinity and intrinsic efficacy
13
Q
What do BZs do
A
- BZs enhance the response to GABA by facilitating the opening of GABA activated chloride channels
- They bind specifically to a regulatory site of the receptor
- act to allosterically increase the affinity of GABA for the receptor
14
Q
What are the different classes of BZs
A
- Bz agonists- 100% efficacy- whether you turn up or down effect of intrinsic GABA
- BZ inverse agonists- bind to the site but produce the opposite effect and are said to have negative intrinsic efficacy- negative allosteric modulators
- BZ antagonists- bind the a/g site but are unable to activate the receptor- intrinsic efficacy=0
15
Q
What are positive allosteric modulators
A
- Full agonist
- Partial agonist
16
Q
What are negative allosteric modulators
A
- Antagonists
- Inverse agonists- turn down amount of chloride going through the GABA receptor
17
Q
What are the pros of BZs
A
- Sedation - pre op
- Anxiolysis
- Hypnotic
- Anticonvulsant
18
Q
What are the cons of BZs
A
- Memory loss
- sedation
- Abuse potential
- Addictive
- Withdrawal symptom
19
Q
Are all GABAARs sensitive to BZs
A
- No
- Only alpha1,2,3 and 5 containing receptors bind BZ with appreciable affinity
- A1,6 are not sensitive
- Beta subunit has no effect on BZ binding
20
Q
What subunit appears to determine BZ effects
A
- Alpha
21
Q
Why was the alpha subunit thought to determine BZ effects
A
- Recombinant GABAAR studies identified that mutating a His to Arg in the BZ binding site abolished binding of classical BZs but does not affect receptor assembly or sensitivity to GABA
- a1, a2, a3 and a5 subunits contain this His residue whereas a4 and a6 contain an Arg residue in the same position
- Sedative and amnesic effects of diazepam are mediated by a1 containing GABAARs
- Anxiolytic effects of diazepam are mediated by a2/a3 containing GABAARs
22
Q
Describe light/dark choice test
A
- His res at position 101 is mutated to Arg In alpha 2 subunit
- Only thing that is changed
- When challenged with BZs – light dark test
- Approach avoidance task
- Amount of time in dark or light compartment changes - no difference in mutated
- Anxiolytics increase time spent in Lit area
- Light is aversive- brightly lit and open, dark is safe and closed
23
Q
Describe the elevated plus test
A
- Different compartments of maze
- Some open and aversive
- Anxiolytics increase time spent in open arms of cross
- When point mutation on alpha 2 there is no effect of BZ on time spent
24
Q
Describe what happens when tests carried out with alpha 3 mutation instead of alpha 2
A
- Same response as without mutation
- Suggests anxiolytic actions of Diazepan can be prescribed to alpha 2 receptors
25
Describe tests carried out to observe locomoter activity
1. Big box with infrared beams across box
2. Number of crossings is measure of locomotive activity
3. Dose dependent decrease in locomotive activity in wildtype and both point mutations of alpha 2 and 3 4. Rorarod- see how long animal takes to fall off a rotating thing – latency to fall decreases with DZ
5. Pentylenetrazole test – seizure induction
6. BZ are anticonvulsant
7. Seen in all animals with point mutation
26
What effects is the alpha 1 subunit responsible for
1. Sedation
2. Amnesia
3. Seizure protection
27
What effect is alpha2 responsible for
1. Anxiolysis
28
What effect is mediated by alpha 3
1. Anxiolysis
29
What effect is mediated by alpha 5
1. Amnesia
30
Describe what happens with point mutations in g2 subunit to BZ agonist effects
1. Knock point mutation in GABAaR gamma 2 subunit
2. Eliminates sensitivity to zolpidem
3. Zolpidem is a hypnotic that is not a BZ but acts at the BZ site
4. Zolpidem is a positive allosteric modulator at the BZ site that potentiates the effects of GABA - which can be detected as increased amplitude and decay times for IPSCs
5. Point mutation of Phe residue to an Ile residue (F77I) in g2 subunit eliminates zolpidem sensitivity electrophysiologically and behaviourally (rotarod)
31
Why is it useful to Identify alcohol actions at specific GABAAR subtypes
1. Alcohol antidotes – acute reversal of alcohol intoxication
2. Alcohol addiction treatments – can you reverse the pleasurable effects of alcohol?
3. Alcohol mimetics – beneficial effects of low dose alcohol – synthetic alcohol
32
What are the CNS effects of alcohol
1. Alcohol is a CNS depressant
2. Apparent stimulatory effects result from depression of inhibitory control mechanisms in the brain
3. Characteristic response: euphoria, impaired thought processes, decreased mechanical efficiency
33
What causes alcohols euphoria and pleasure effects
1. Dopamine and opioids receptor
34
What causes alcohols anxiolysis effects
1. Increase in GABA
35
What causes alcohols sedation/amnesia effects
1. Increase in GABA
2. Decrease in NMDA
36
What causes alcohols nausea
1. 5HT3
37
What causes alcohols neuroadaptation
1. NMDA. 5HT
38
What causes alcohols stress effects
1. CRF receptor
39
What casues alcohols withdrawal effects
1. GABA
2. NMDA
40
What is alcohol to GABAARs
1. Positive allosteric modulator
41
Describe effect of alcohols at GABAARs
1. Effects of alcohol similar to that of barbiturates and BZs
2. BZs widely used to treat effects of abrupt alcohol withdrawal (seizure)
3. High [ethanol] (~100mM) can potentiate GABA responses at most GABAA receptors
4. a4/a6, b, d containing GABAARs are sensitive to ethanol (threshold response at 3mM) – one glass of wine – subunits found extrasynaptically
5. a4/a6, b, d containing GABAARs are sensitive to low concentrations of GABA (nM) and show little desensitization
6. Ethanol enhances GABA mediated tonic inhibition
42
What does Ro15-4513 do
1. Ro 15-4513 is a behavioural alcohol antagonist
2. Efficacy in reducing alcohol drinking in rodents
3. Structural related inverse agonists RY080 and RY024 also show behavioural antagonist actions
4. Ro15-4513 and related show high affinity for a4/a6,b,d containing receptors
5. Not all BZ inverse agonists show alcohol antagonism so where is the binding site? Is it the BZ site?
6. Alcohol can displace Ro15-4513 in binding assays so share a (overlapping) binding site
7. Analagous to H101 in a1 subunits is a6 R100 – R100Q mutation enhances alcohol sensitivity to alcohol
8. Where arg residue is mutated and you lose alcohol sensitivity
9. binding site is homologous but different to BZ site.
10 .BZ don’t bind to alpha4, alpha 6 but binding pocket is similar?
