Lec 6- GABA

Question 1

Synthesis of GABA and glycine

Answer 1

• GABA is formed from glutamate by enzyme Glutamic acid decarboxylase GAD
• All enzymes that release GABA contain GAD
• GABA is broken down by enzyme GABA-T (GABA transaminase)
• Synthesis and metabolism linked to Kreb cycle

Question 2

Synthesis and metabolism of GABA: I

Answer 2

• GABA is synthesised from glutamate by the enzyme glutamic acid decarboxylase (GAD)
• GAD is found only in those neurons which synthesise GABA and thus the location is a valuable identifier of GABAergic neurons
• GABA is metabolised by the ubiquitous enzyme GABA transaminase
• GABA is release by interneurons- released locally to act on neurones close to the release
• GAD is anaerobic while GABA transaminase is aerobic thus after death the levels of GABA in the brain increase very rapidly
• Inhibitors of GAD will cause a decrease in the levels of brain GABA and cause convulsions
• Several inhibitors of GABA transaminase have been developed which lead to increases in the levels of brain GABA; these compounds are anti-convulsants

Question 3

Storage and release of GABA and glycine

Answer 3

• Both GABA and glycine appear to be stored in synaptic vesicles and their release is by Ca²⁺ dependent exocytosis
• Both GABA and glycine are removed from the synaptic cleft by active re-uptake processes
  
  • This is the means by which their actions are terminated subsequent to their release

Question 4

Reuptake of GABA and glycine

Answer 4

• Both GABA and glycine are removed from the synaptic cleft by active reuptake processes
• This is the means by which their actions are terminated subsequent to their release

Question 5

Inhibitory amino acid receptors

Answer 5

• Glycine receptors are ionotropic receptors
• GABA receptors are of 2 types:
  
  • GABA_A receptors are ionotropic- allow ions to pass (Cl^-), directly linked to ion channels
  • GABA_B receptors are metabotropic- GPCR linked to 2nd messengers= change the metabolism of cell= Much slower due to cascade

Question 6

Glycine receptors

Answer 6

• The glycine receptor is an ionotropic receptor and is closely related structurally to the GABA_A receptor
• Glycine receptors are found mainly in the spinal cord; the convulsant drug strychnine (death by asphyxiation- muscle contraction) is a competitive glycine antagonist
• The channel is anion specific
• The functional receptor is composed of 5 subunits which surround the ion channel; there are 3 alpha and 2 beta subunits in the adult receptor
• The natural agonist is glycine but the receptor may also be activated by beta-alanine and taurine
• Strychnine is the most potent selective antagonist with affinities in the range of 5-15 nM

Question 7

GABA_A and GABA_B receptors -EXAM

Answer 7

• GABA_A receptors are found mainly post-synaptically and are directly coupled to chloride channels the opening of which reduces neuronal excitability; the convulsant bicuculline (convulsant) is a competitive antagonist, picrotoxin blocks GABA_A receptor channel
• GABA_B receptors are mainly responsible for pre-synaptic inhibition and are coupled to 2^nd messengers
• Baclofen is an agonist
• Antagonist include phaclofen, 2-OH, saclofen
• Can cause subsequent inhibition of neurotransmitter- If GABA = autoreceptor- it does this by blocking Ca2+= stop release of Ca2+ dependent exocytosis

Question 8

Classification of GABA receptors- GABA_A effects

Answer 8

• Post-synaptic inhibition
• Increase Cl^- conductance

Question 9

Classification of GABA receptors-GABA_A Agonist

Answer 9

• GABA = +
• Muscimol = +
• Baclofen = -

Question 10

Classification of GABA receptors- GABA_A Antagonists

Answer 10

• Bicuculline= +
• Picrotoxin= +
• Phaclofen= -

Question 11

Classification of GABA receptors- GABA_B Effects

Answer 11

• Pre-synaptic inhibition
• Decreased Ca²⁺ conductance
• Increase in K⁺ conductance

Question 12

Classification of GABA receptors- GABA_B Agonist

Answer 12

• GABA= +
• Muscimol= -
• Baclofen= +

Question 13

Classification of GABA receptors- GABA_B Antagonists

Answer 13

• Bicuculline= -
• Picrotoxin= -
• Phaclofen= +

Question 14

Comparison of the distribution of GABA_A and GABA_B receptors in the cerebellum

Answer 14

Question 15

GABA_B receptor: I

Answer 15

• GABA_B receptors are metabotropic receptors being coupled to second messenger systems
• Activation of the receptors located on pre-synaptic terminals suppress the release of neurotransmitter by inhibition of inward Ca²⁺ conductance
• Activation of the receptors located post-synaptically produces a long-lasting hyperpolarisation by increasing K+ conductance
• The functional receptor is composed of 2 protein subunits: GABA_BR1 + 2
• These proteins are 7 transmembrane proteins with amino acid sequence homology of about 35%
• The distribution of the 2 proteins appears to be similar and it appears that both are expressed together to provide the pharmacology that we expect of GABA_B receptors in vivo
• This is the only G-protein coupled a receptor presently known that functions as a heterodimer; subunit associate via their C-termini

Question 16

GABA_B receptors in the CNS

Answer 16

• GABA_B receptors may be involved in
  
  • Control of epilepsy
  • Regulation of the hypothalamic-pituitary axis
  • Enhancement of anti-depressant drug action
  • Suppression of cocaine withdrawal symptoms
  • Alleviation of pain

Question 17

GABA_A receptors in the CNS- GABA_A receptors are involved in

Answer 17

• Sedation
• Anxiety
• Muscle relaxation
• Convulsive activity
• Amnesia making midazolam in particular a valuable pre-anaesthetic

Question 18

GABA_A receptors: I

Answer 18

• Major inhibitory amino acid receptor in the brain
• Channel is anion specific (Cl^-)
• GABA activation causes an inhibitory post-synaptic potential (IPSP)
• Agonists: GABA, muscimol
• Antagonist: bicuculline
• Channel blocker: Picrotoxin
• Change of chloride gradient occurs in both early development and epilepsy

Question 19

GABA_A receptors: II - GABA_A receptors also have binding sites for

Answer 19

• Benzodiazepines- diazepam
• Barbiturates
• Neurosteroids- alfoxalone (anaesthetic)

Question 20

GABA_A receptor- additional binding site

Answer 20

• Benzodiazepines
  
  • potentiate the action of GABA by shifting the GABA dose-response curve to left- (Same amount of GABA = greater response)
  • Increase frequency of channel opening
• Barbiturates
  
  • Potentiate actions of GABA by shifting the dose-response curve to the left and increasing the maximum response
  • Increase channel open lifetime
• Steriods
  
  • Potentiate actions of GABA
  • Increase channel opening frequency and open lifetime

Question 21

Allosteric modulation of the GABA_A receptors

Answer 21

• A-is the theoretical dose-response curve which induces an increased chloride conductance as the concentration of GABA is increased
• B- represents the leftward shift in that dose-response curve which would be seen in the presence of a benzodiazepine agonist
• C- represents the leftward shift and increased maximum induced by pentobarbitone

Question 22

Accessory site agonist of GABA_A receptors

Answer 22

Question 23

Evidence to support this mechanism of action

Answer 23

• There is a strong correlation between the affinity of individual agonist benzodiazepines for their binding site in the brain and the therapeutic dose required to produce their overt effects
• Flumazenil (Anexate) acts as an antagonist at the benzodiazepine site and blocks the effects of both the agonist and inverse agonist benzodiazepines
• The effects of the benzodiazepine agonist are directly correlated with blood levels of the drug; the relief of anxiety is immediate

Question 24

Inverse agonist at the benzodiazepine site

Answer 24

• In 1980 a compound, ethyl b-carboline-3-carboxylate (b-CCE) was discovered that competitively displaced the benzodiazepines from their binding sites in brain membranes
• Behavioural experiments showed that this compound had effects diametrically opposed to those of the classical benzodiazepines: these compounds are now referred to as inverse agonist

Question 25

Efficacy spectrum of benzodiazepine receptor site ligand

Answer 25

Question 26

Structure of GABA_A receptor

Answer 26

Question 27

Cloning of GABA_A receptor subunits

Answer 27

• Several classes of GABA_A receptor subunit have been cloned
  
  • Alpha subunit of which there are 6 isoforms
  • Beta of which there are 3 isoforms
  • Gamma subunit of which there are 3 isoforms- the gamma unit is located within the synapse itself
    
    • Delta= extrasynaptic location
• Functional receptors are made up of 5 subunits arranged pseudosymmetrically around the integral ion channel
• The most common GABA_A receptor of the mammalian brain consists of 2X a1, 2X b2 and 1X y2 subunits
• The different sub-units, give slightly different physiological responses

Question 28

GABA_A receptor subunit isoform

Answer 28

Question 29

Why all these subunits

Answer 29

• The functional characteristics of a particular GABA_A receptor are defined by its precise subunit composition
• The alpha subunit is important in the recognition of the benzodiazepines
• The b-subunits are important in the recognition of the natural agonist GABA- must have 2 units
• The y subunit is an important structural subunit

Question 30

Synthesis and metabolism of GABA: II

Answer 30

• GAB in anaerboic while GABA transaminase is aerobic thus after death the levels of GABA in the brian increase very rapidly

Question 31

Different Alpha units = different actions

Answer 31

• Alpha-1= sedative- Mainly in the cerebellum, hippocampus, globus Palabras
• Alpha 2,3= Anxiolytic- 2= hippocampus ; 3= Amygdala
• Alpha 5= striatum
• Alpha 5= CA1, CA3
• Alpha 6= cerebellum
• These are dynamic, they change during time, pathology (epilepsy), hormonal cycles