Inhibitory Amino Acids (A*)

Question 1

List the 3 major inhibitory amino acids.

Where in the nervous system are they located?

Which of these are able to cross the blood-brain barrier?

Answer 1

Major inhibitory amino acids include:

1 - GABA (the principal inhibitory amino acid in the CNS).

2 - Glycine (localised mostly to the spinal cord and brainstem, where it is the principal inhibitory neurotransmitter).

3 - Taurine (found in all areas of the CNS).

• None of these are able to cross the blood brain barrier.

Question 2

In which type of neurones is GABA mainly found?

Answer 2

GABA is mainly found in interneurones.

Question 3

With which other molecule is GABA colocalised?

Answer 3

GABA is colocalised with its synthesising enzyme, GAD.

Question 4

Give a brief overview of GABA metabolism.

What is the purpose of GABA metabolism?

Answer 4

1 - GABA is moved to glial cells.

2 - Here, GABA-T and SSADH convert GABA to succinic acid.

3 - Succinic acid is shunted into the mitochondria, where it is used in the krebs cycle to produce glutamine.

4 - Glutamine is moved back to GABAergic neurones.

5 - Here, glutamine dehydrogenase converts glutamine into glutamate.

6 - Then, GAD decarboxylates glutamate to form GABA.

• The cycle repeats.
• GABA metabolism is a way of controlling the level of GABA activity.

Question 5

List the types of GABA receptors.

Are these ionotropic or metabotropic?

Answer 5

Types of GABA receptors include:

1 - GABAA (ionotropic).

2 - GABAB (metabotropic).

3 - GABAC (ionotropic).

Question 6

Describe the process that terminates the activity of GABA at a synapse.

Answer 6

• Termination of GABA activity at a synapse occurs by breakdown of the receptor-GABA complex and reuptake of GABA.
• GABA reuptake can occur at presynaptic neurones or glia via GABA transporters:

1 - GAT1 transporters on the presynaptic membrane.

2 - GAT2/GAT3 transporters on glia that are in close proximity to the synapse.

Question 7

What is the function of the vesicular GABA transporter (vGAT) and other vesicular inhibitory amino acid transporters?

Answer 7

• Excess GABA can be stored in vesicles for future release.
• The vesicular GABA transporter (vGAT) loads GABA into vesicles for storage and exocytosis for neurotransmitter release.
• There are similar transporters for other inhibitory amino acids such as glycine for the same purpose.

Question 8

Give an overview of the GABAA receptor.

Include:

1 - The type of receptor.

2 - The localisation of the receptor.

3 - The mechanism by which it produces inhibitory postsynaptic potentials.

4 - A brief description of its structure.

5 - 2 agonists (other than GABA).

6 - 3 antagonists.

7 - 4 positive modulators.

8 - A GABAA channel blocker.

Answer 8

1 - GABAA is an ionotropic receptor (ligand-gated ion channel).

2 - GABAA is widespread in the CNS in postsynaptic terminals of interneurones.

3 - GABAA produces fast inhibitory postsynaptic potentials by increasing Cl- conductance.

4 - GABAA is a pentamer (has 5 subunits), containing 2 alpha subunits, 2 beta subunits and 1 gamma subunit. There is a Cl- channel at the centre of the subunits.

5 - Agonists of GABAA receptors other than GABA include muscimol and general anaesthetics.

6 - Antagonists of GABAA receptors include bicuculline, flumazenil and gabazine.

7 - Positive modulators include barbiturates, benzodiazepines, ethanol and neurosteroids.

8 - A GABAA channel blocker is picrotoxin.

Question 9

Give an overview of the GABAB receptor.

Include:

1 - The type of receptor.

2 - The localisation of the receptor.

3 - The mechanism by which it produces inhibitory postsynaptic potentials.

4 - A brief description of its structure.

5 - 1 agonist (other than GABA).

6 - 1 antagonist.

Answer 9

1 - GABAB is a metabotropic receptor (GPCR).

2 - GABAB is widespread in the CNS in both postsynaptic and presynaptic terminals of interneurones.

3 - GABAB is coupled to Gi/o. It produces presynaptic inhibition by inhibiting adenylyl cyclase (and therefore decreasing cAMP, which in turn decreases Ca2+ influx) and produces postsynaptic inhibition by increasing K+ influx (explained in more detail in a later card). These IPSPs are slow compared to GABAA.

4 - GABAB is a dimer (has 2 subunits). It is a GPCR.

5 - An agonist for GABAB is baclofen.

6 - An antagonist for GABAB is phaclofen.

Question 10

How many domains span the subunits of GABAA?

Answer 10

Each of the 5 subunits of GABAA (2x alpha, 2x beta and 1x gamma) contain 4 transmembrane domains (M1-M4).

Question 11

To which type of GABA receptor does benzodiazepine bind?

Which subunits are required for benzodiazepine to bind to this type of GABA receptor?

Answer 11

• Benzodiazepine binds to GABAA receptors.
• Benzodiazepine binding requires an alpha and gamma subunit, which only exist on GABAA receptors (not GABAC, which consists of 5 rho subunits).

Question 12

What effect do benzodiazepines, barbiturates, neurosteroids and ethanol have on GABAA receptors?

Briefly outline the mechanism by which these drugs do this.

What is the clinical effect of these drugs?

Answer 12

• Benzodiazepine, barbiturates, neurosteroids and ethanol increase GABA transmission by exerting a neuromodulatory effect on GABAA receptors.
• Benzodiazepines do this at the benzodiazepine binding site by increasing the frequency of channel openings by increasing the GABAA receptor affinity for GABA.
• Barbiturates do this at the barbiturate binding site by favouring the longest channel open state (9ms).
• Neurosteroids do this at the Cl- channel by modulation of benzodiazepine binding and by enhancing agonist binding to the GABA site.
• Ethanol does this at the ethanol binding site by increasing both the frequency and duration of channel opening.
• Clinically, this results in anxiolytic and sedative effects.

Question 13

At which site on the GABAA receptor does flumazenil act?

What is its effect here?

How is it used clinically?

Answer 13

• Flumazenil competitively inhibits the activity of benzodiazepines at the benzodiazepine binding site on the GABAA receptor.
• Clinically, it is used to reverse the effects of sedation.

Question 14

List 3 clinical effects of GABAA receptor activation.

Give examples of drugs that can achieve each effect.

Answer 14

GABAA receptor activation can cause:

1 - Anticonvulsant effect.

Direct action:

• Phenobarbitone.
• Benzodiazepines.

Indirect action:

• Tiagabine (GAT-1 inhibition).
• Vigabatrin (GABA-T inhibition)

2 - Anxiolytic effect.

• Benzodiazepines.

3 - General anaesthetic affect.

• Neurosteroids such as alphaxalone.
• There are other clinical effects that are not discussed here.

Question 15

What is the major GABAA receptor subtype?

What is the second most common GABAA receptor subtype?

How does the clinical effect of benzodiazepine differ when acting at each of these receptors?

Answer 15

• Alpha 1 beta 2 gamma 2 (these are the subunit types) is the major subtype of GABAA receptors, and represents ~60% of all GABAA receptors.
• This subtype is responsible for the sedative, amnesic and anticonvulsant actions of benzodiazepines.
• Alpha 2 beta 3 gamma 2 (these are the subunit types - there are still only 5 of them) is the second most common GABAA receptor subtype, and represents ~20% of all GABAA receptors.
• This subtype is responsible for the anxiolytic action of benzodiazepines.

Question 16

What is the clinical use of zolpidem?

How does it work?

Answer 16

• Zolpidem is a hypnotic drug.

- It is a GABAA agonist that has a particularly high affinity for the alpha 1 subunit.

Question 17

How many GABAB receptor subtypes are there?

Answer 17

There are 2 GABAB receptors: GABAB 1 and GABAB 2.

*Unlike GABAB, there are many subtypes of GABAA receptors.

Question 18

Why are the GABAB receptor subtypes often coexpressed?

Answer 18

• The 2 GABAB receptor subtypes are often coexpressed because they have a synergistic effect:
• GABAB 1 facilitates the coupling of the GABAB 2 receptor to its G protein.
• GABAB 2 increases the affinity of GABAB 1 for GABA.

Question 19

Describe the downstream pathways and resulting effects of the of the GABAB GPCR.

Answer 19

If the GABAB receptor is located on the presynaptic terminal (an autoreceptor):

1 - Activation of the GABAB receptor causes a reduction in intracellular cAMP by inhibiting adenylyl cyclase.

2 - This causes increased closure of voltage-gated Ca2+ channels.

3 - This decreases neurotransmitter release at the presynaptic terminal.

If the GABAB receptor is located on the postsynaptic terminal:

1 - Activation of the GABAB receptor causes an increase in K+ conductance via effect on the GIRK and TREK2 potassium channels.

2 - This results in an increase in K+ efflux.

3 - This causes hyperpolarisation, and generates a slow IPSP.

Question 20

What is the most clinically used GABA-B receptor agonist?

What is it used for?

Answer 20

• The most clinically used GABA-B receptor agonist is baclofen.
• It can be used to treat:

1 - Spasticity.

2 - Pain.

3 - Migraines.

4 - Addiction.

Question 21

What is the precursor to glycine?

Which enzyme converts this into glycine?

Answer 21

• L-serine is the precursor to glycine.

- Serine hydroxymethylase converts L-serine into glycine.

Question 22

What type of receptor is the glycine receptor?

Answer 22

• Glycine is an ionotropic receptor.

- It is a Cl- channel.

Question 23

List 3 agonists for glycine receptors (other than glycine).

List 1 antagonist for glycine receptors.

Answer 23

Glycine receptor agonists (other than glycine):

1 - Alanine.

2 - Taurine.

3 - Proline.

Glycine receptor antagonist:

1 - Strychnine.

Question 24

What are the signs of strychnine poisoning?

Answer 24

Strychnine poisoning is characterised by:

1 - Spinal overexcitation.

2 - Muscle spasms.

3 - Convulsions.

Question 25

What is hyperekplexia?

What causes it?

Answer 25

• Hyperekplexia is an exaggerated startle response.

- It is caused by a mutation to the alpha 1 subunit of the glycine receptor.

Question 26

Describe the structure of the glycine receptor.

Where on the glycine receptor do glycine and strychnine bind?

Answer 26

• The glycine receptor is pentameric (has 5 subunits) like the GABAA receptor.
• It contains 3 alpha subunits and 2 beta subunits.
• There are 4 subtypes of the alpha subunit (alpha 1-4).
• Each subunit contains 4 alpha helix transmembrane domains (TM1-4).
• There is a Cl- channel in the centre of the subunits which is lined by the second transmembrane domain (TM2) of each receptor subunit.
• The large extracellular domain on the alpha subunits is the binding site for glycine and strychnine.

Question 27

What are the functional differences in the subtypes of alpha subunits in the glycine receptor?

Answer 27

• Alpha 1 subunits are involved in glycine receptors that influence motor control.
• Alpha 2 subunits are strongly expressed in embryonic neurones, but decline in adulthood.
• Alpha 3 subunits are involved in glycine receptors that influence pain sensation.
• Who knows about alpha 4 subunits.

Question 28

What is gephyrin?

What is its function?

Answer 28

• Gephyrin is a protein that anchors glycine receptors to the postsynaptic membrane.
• Gephyrin does this by interacting with actin-binding protein and profilin in order to link the glycine receptors to the microtubules of the cytoskeleton.

Question 29

Describe the process that terminates the activity of glycine at a synapse.

Give an example of a pharmacological agent that targets this process.

Answer 29

• Termination of glycine activity at a synapse occurs by breakdown of the receptor-glycine complex and reuptake of glycine.
• Glycine reuptake can occur at neurones or glia through glycine transporters:

1 - GLYT-1 is expressed in both glia and neurones.

• GLYT-1 is sensitive to sarcosine, which is a glycine reuptake inhibitor used to treat symptoms of schizophrenia and depression.

2 - GLYT-2 is mainly expressed in neurones.

Question 30

Describe the process of Cl- homeostasis in neurones expressing GABAA.

Answer 30

• A Na+/K+ ATPase pumps 3 Na+ out of the neurone for every 2 K+ in.
• This raises intracellular [K+].
• A KCC2 cotransporter transports both K+ and Cl- out of the cell in equal ratios.
• The KCC2 cotransporter raises both extracellular K+ (for ATPase activity) and extracellular Cl- (for GABAA receptor activity).

Question 31

How does GABAA transmission differ in immature neurones?

Why is this important?

Answer 31

• In immature neurones, Cl- homeostasis works differently:
• Immature neurones weakly express the KCC2 cotransporter, which is important in maintaining high extracellular Cl- for the GABAA receptor (which is meant to cause Cl- influx).
• Furthermore, immature neurones express NKCC1, which cotransports Na+, K+ and Cl- into the neurone (Na+ influx driven by the Na+/K+ ATPase).
• Therefore, in an immature neurone, the Na+/K+ pump drives Cl- influx via NKCC1 (whereas in a mature neurone, the Na+/K+ pump drives Cl- efflux via KCC2).
• This results in high intracellular Cl-, so when the GABAA receptor opens, Cl- efflux occurs rather than Cl- influx.
• GABAA therefore causes depolarisation rather than hyperpolarisation in immature neurones.
• This is important because if a GABAergic anticonvulsant drug affects immature GABAergic neurones, it will actually be epileptogenic.

Question 32

A*:

List 5 neuroprotective functions of GABA.

Answer 32

Neuroprotective functions of GABA include protecting against:

1 - Neurodegeneration.

2 - Sleeplessness.

3 - Depression.

4 - Cognitive impairment.

5 - Memory loss.

Question 33

A*: List 7 non-neurological diseases for which GABA supplements can be used as a preventative therapy.

Answer 33

Non-neurological disorders for which GABA supplements can be used as a preventative therapy:

1 - Hypertension (explained in more details in a later card).

2 - Diabetes (explained in more details in a later card).

3 - Cancer (explained in more details in a later card).

4 - Allergy.

5 - Hepatic diseases.

6 - Renal diseases.

7 - Intestinal diseases.

Question 34

A*:

List 2 possible mechanisms by which GABA reduces hypertension.

Answer 34

Possible mechanisms by which GABA reduces reduced hypertension include:

1 - Inhibition of sympathetic vasoconstrictor neurones by presynaptic GABAB receptors.

2 - Inhibition of ACE by GABA, reducing angiotensin II and therefore reducing vasoconstriction.

Question 35

A*:

List 2 possible mechanisms for the antidiabetic effect of GABA.

Answer 35

Possible mechanisms for the antidiabetic effect of GABA:

1 - GABA increases Ca2+ influx in islet cells (alpha, beta and delta), which in turn stimulates growth and survival by activating the PI3K/Akt pathway.

2 - GABA stimulates beta cell proliferation by upregulating the transcription of hepacam 2, urocortin 3 and wnt4.

Question 36

A*:

List 3 possible mechanisms for the anti-cancer effect of GABA.

Give an example of a possible mechanism for the oncogenic effect of GABA.

Answer 36

Possibles mechanisms for the anti-cancer effect of GABA:

1 - GABA inhibits matrix metalloproteinase function, suggesting GABA prevents tumour metastasis because MMPs are usually involved in metastasis as they degrade the tumour surface and adhesion sites.

2 - GABA prevents cell cycle progression, for example by inhibiting expression of EGR1, a transcription factor that plays a role in cell proliferation and differentiation (GABA prevents tumorigenesis).

3 - GABA inhibits VEGF, preventing tumour vascularisation.

Possible mechanism for the oncogenic effect of GABA:

• GABA inhibits caspase enzymes, which promote apoptosis.
• This might explain why a GABAB receptor antagonist, CGP, was recently shown to have an anti-cancer effect (Kanbara et al., 2018).

Question 37

A*:

List 4 behaviours of the brain that involve GABAergic transmission.

Answer 37

Behaviours of the brain that involve GABAergic transmission:

1 - Fear and anxiety.

2 - Learning and Memory.

3 - Reward and addiction.

4 - Stress and depression.

*This is not an exhaustive list.

Question 38

A*:

Describe the organisation of GABAergic neurones in the amygdala and how this contributes to fear and anxiety behaviour.

Which other regions of the brain that are involved in fear and anxiety behaviour contain GABAergic neurones?

Answer 38

Organisation of GABAergic neurones in the amygdala:

• GABA is involved in 4 of the 10 nuclei of the amygdala: the lateral and basal (basolateral), centrolateral and centromedial (central) nuclei. This GABAergic network is largely mediated by GABAA receptors.
• The lateral and basal nuclei consist of many non-GABAergic excitatory neurones and a lesser number of GABAergic interneurones.
• In the central nuclei, GABAergic neurones are both the principal neurones and interneurones.
• The central and basolateral nuclei are connected by another mass of GABAergic interneurones, providing a means of feedback between the nuclei.
• It was shown that the activity of GABAergic neurones in the centrolateral nucleus increased during fear responses (Haubensak et al., 2010). The same neurones receive a tonic inhibition in the absence of a fear-inducing stimulus. Knockdown of alpha5 GABAA receptors in these neurones leads to spontaneous firing of these neurones, causing increased anxiety (Botta et al., 2015). Alpha5 GABAA receptor agonists may therefore have potential for producing anxiolysis.
• Other regions of the brain that are involved in the fear response that contain GABAergic neurones include:

1 - Hippocampus.

2 - Prefrontal cortex.

3 - Periaqueductal grey area.

4 - Locus coeruleus.

Question 39

A*:

Which GABA-targeting drug is known to have an impact on learning and memory?

Describe the organisation of GABAergic neurones in the hippocampus and how this contributes to learning and memory.

Answer 39

• The amnesia-inducing effects of benzodiazepines indicate that GABAA receptors inhibit learning and memory.
• Alpha5 GABAA receptors are expressed in both the dorsal and ventral hippocampus, but more so in the ventral.
• It is generally considered that the dorsal hippocampus is involved in learning and memory whereas the ventral hippocampus is involved in emotional processing. However, in the ventral hippocampus, alpha5 GABAA receptor activity increases the LTP threshold, reducing synaptic plasticity and therefore memory formation.
• Knockdown of alpha5 GABAA receptors improves learning and memory in mice (Soh et al., 2015).
• Alpha5 GABAA receptor antagonists have been shown to improve cognitive function in disorders such as Down syndrome and schizophrenia (Rudolph et al., 2014).

Question 40

A*:

Describe a mechanism for non-canonical dopamine-gaba corelease.

Where does this occur?

Answer 40

• Some dopaminergic neurones in the corpus striatum express the GABA-synthesising enzyme, GAD 65.
• These neurones are able to induce a GABA-mediated inhibitory potential, with concomitant dopamine release.
• The process of GABA transport in these neurones is considered ‘non-canonical’ because removal of the VGATs that normally load GABA into vesicles for exocytosis has no effect on the generation of GABA-induced postsynaptic potentials.
• On the other hand, disabling VMATs prevents the inhibitory postsynaptic potential, suggesting that GABA is loaded into vesicles by VMATs rather than VGATs in striatal neurones (Tritsch et al., 2012).