GABAA Receptors,Anxiolytics, Sedatives,

Hypnotics

Outline• Physiology of GABAA responses• How is GABA synthesized, released and metabolized?

– Vitamin B6– Stiff-man syndrome

• Pharmacology:– Agonists, competitive & non-competitive antagonists: How to

study?

• Sedatives, anxiolytics & hypnotics • Benzodiazepine agonists, competitive antagonists,

inverse agonists (allosteric modulators)• Next lecture: How to identify the GABA receptor subunits

that are responsible for the different effects of benzodiazepines

-Aminobutyric Acid (GABA)

• GABA occurs in brain tissue, trace amount in other tissue

• GABA functions as an inhibitory transmitter in many different CNS pathways

• 20% of CNS neurons are GABAnergic– Most are short interneurons– But long GABAnergic tracts run to cerebellum and

striatum• GABA serves as a transmitter at about 30-40% of all

the synapses in the CNS

AP may be initiated AP not initiated. AP less likely to be initiated

Synthetic enzyme:Glutamic acid decarboxylase (GAD)

Co-factor:Vitamin B6 (pyridoxalphosphate)

What happens if insufficient GABA is synthesized?• Stiff-man syndrome• Or Vitamin B6 deficiency

GABA Receptors • GABA acts on two distinct types of receptor

– GABAA receptor: ligand-gated channel– GABAB receptor:GPCR

• GABAA receptors – located postsynaptically– Serves as main fast inhibitory neurotransmitter in the brain– Selectively permeable to Cl- ,hyperpolarizes membrane & inhibits

action potential firing

• GABAB receptors – located pre- and postsynaptically– GPCR; functional receptor is dimer– Inhibitory action– Stimulates opening of K+ channels & inhibits voltage-gated Ca2+

channels

GABAA Receptor• 19 subunits encoded by 19 distinct

genes – 1-6, 1-3, 1-3, 1-2– Selectively permeable to Cl-

• Each subunit– 4 transmembrane segments – Both amino and carboxyl termini

extracellular – These extracellular segments form the

binding sites for GABA and for allosteric modulators

• Member of the Cys-loop superfamily(related to nAChR and Glycine R)

GABAA Receptor: Phasic vs. Tonic

Neurotherapeutics. 2007, 4:18

Phasic inhibition• at synapse• Fast transient inhibitory postsynaptic

currents due to synaptically released GABA• α2β2γ subunits• Benzodiazepine sensitive

Tonic inhibition• at extrasynaptic sites• Responds to background GABA

concentrations• α2β2δ subunits• Benzodiazepine insensitive

Competitive Antagonist

Noncompetitive Antagonist

Agonist

GABA Antagonists Cause Convulsions!

No change in total number of binding sites [Rt].

Decrease in apparent agonist affinity (increase in Kd).

Competitive Inhibition

Decrease in # functional receptors or decrease in apparent # of available agonist binding sites [Rt].

No change in apparent affinity (no change in Kd).

Non-competitive Inhibition Many ways to interact with Receptors

Drugs acting on GABAA Receptors

CNS Neurol Disord Drug Targets. 2008, 7:211

Agonists & Antagonists: Benzodiazepines:

Pore Blockers:

Other Allosteric Modulators:

β-carbolines

• Agonists– GABA: Endogenous agonist– Muscimol: derived from hallucinogenic mushroom, resembles

GABA chemically, powerful GABAA receptor agonist– Gaboxadol: synthetic analogue, partial agonist

• Antagonists– Bicuculline: naturally occurring convulsant compound, is a

specific antagonist that blocks fast inhibitory synaptic potential in most CNS synapses

– Gabazine: synthetic GABA analogue

• Channel blocker– picrotoxin: convulsant, acts by blocking Cl- channel pore of

the GABAA receptor, thus blocking the postsynaptic inhibitory effect of GABA

Drugs acting on GABAA Receptors

• Benzodiazepines– Four among the 200 most prescribed drugs in US (alprazolam (Xantax),

clonazepam (Klonopin), diazepam (Valium), lorazepam (Ativan))– Selectively potentiate the effects of GABA on GABAA receptors– Bind with high affinity to a separate site on GABAA receptor that is

different from the agonist binding site for GABA – Binding of GABA is facilitated and its agonist effect is enhanced.– Not all GABAA receptors are affected by benzodiazepines– Sedative benzodiazepines such as diazepam (Valium) are benz agonists– Convulsant analogues: flumazenil is a benzodiazepine antagonist; β-

carbolines are benzodiazepine inverse agonists• Barbiturates

– CNS depressant• Anesthetic agents and neurosteroids

– IV anesthetic for fast-acting induction of anesthesia—propofol, etomidate, potentiate the effect of GABA

– Neurosteroids: allosterically enhance activation of GABAA R, as well as activating conventional intracellular steroid receptors

Allosteric Modulators of GABAA Receptors Anxiolytics, Sedatives, Hypnotics• Anxiolytics

– Reduce anxiety– Short-term relief of agitation due to fear or stress, preferably

with minimal CNS depression

• Sedatives– Induce sedation, exert calming effect

• Hypnotics– Produce drowsiness– Encourage onset and maintenance of sleep

diazepam

Anxiolytics

• Benzodiazepines

• Barbiturates

• 5-HT1A agonists

• also β-adrenergic receptor antagonists

Normal Sleep Rhythm

Hypnotics:Effect of long-term

benzodiazepine treatment on sleep quality

• Benzodiazepines decrease time to fall asleep and decrease brief waking periods during sleep,

But …

• Benzodiazepines alter sleep architecture

• Change stages of sleep; less deep slow-wave sleep

• Can cause tolerance• Cause rebound insomnia when

discontinued

Barbiturates have a low margin of safety

Benzodiazepines increase the response to GABA; Benzodiazepine inverse agonists decrease response to GABA

Benzodiazepines increase GABA binding affinity

Resulting in an increased response for the same concentration of GABA

Benzodiazepines have multiple effects:• Anti-anxiety• Sedative• Block memory

– e.g. Rohypnol (flunitrazepam):• (Don't accept drinks from other people. • Keep your drink with you at all times• Have a friend with you)

• Muscle relaxation, cause physical clumsiness• Anti-convulsive• Addiction

Are these due to the same or different receptors?Goal: make selective drugs to a specific receptor target

Summary• GABAA receptors are Cl- channels that produce inhibitory responses

in the CNS: primarily in brain, secondarily in spinal cord

• Glutamate decarboxylase (GAD) requires pyridoxal phosphate (derived from Vitamin B6): deficiency in formula causes seizures

• Autoimmunity to GAD causes stiff-man syndrome

• Agonists: GABA, muscimol. Hill plot indicates 2 agonists bind.

• Competitive antagonist: bicuculline (decrease apparent affinity (increase Kd), no change in # binding sites) cause convulsions

• Non-competitive antagonist: picrotoxin (fish poison), dieldrin (insecticide) bind to pore & block pore (No change in Kd, decrease apparent # receptors) cause convulsions

GABAA Receptors,Anxiolytics, Sedatives,

Hypnotics_________________

Glycine Receptors

Benzodiazepines have multiple effects:• Anti-anxiety• Sedative• Block memory

– e.g. Rohypnol (flunitrazepam):• (Don't accept drinks from other people. • Keep your drink with you at all times• Have a friend with you)

• Muscle relaxation, cause physical clumsiness• Anti-convulsive• Addiction

Are these due to the same or different receptors?Goal: make selective drugs to a specific receptor target

GABAA Receptor(or 5 in retina)

Pentameric, each subunit ~50 kDa

GABA, agonists, competitive antagonists (bicuculline): 2 sites at - interfaces

Non-competitive antagonists (picrotoxin, dieldrin): block pore

Benz-sensitive receptors contain:or and

Benzodiazepines: - interface

Can a non-sedating benzodiazepine be developed that retains anxiolytic and/or anticonvulsant effects?

Goal: Identify receptor subtypes for anxiolytic, anti-convulsant and hypnotic effects.

Strategy to Identify Benzodiazepine Receptor with anxiolytic function:

Make diazepam-insensitive subunits.Is anxiety still reduced by diazepam?

Low et al. (2000) Molecular and Neuronal Substrate for the Selective Attention of Anxiety, Science 290, 131-4

Creating a “Knock-in” Mouse

Low et al. (2000)

Are protein levels normal for the mutant animals?•Yes, determined by immunoblot.

Are the mutant GABA receptor subunits expressed in the correct tissues?•Compare immunocytochemical localization of Ro15-4513 (benzodiazepam inverse agonist that binds normally) + diazepam

What is the physiological response of the mutant GABA receptors?

•Response to diazepam is reduced as expected.

Low et al. (2000)

Behavioral tests:• Sedation (locomotor activity)

• Balance (rotorod test)

• Anti-convulsive effects (phentylenetetrazole test)

• Anxiety (enter lighted area)

• Anxiety (elevated maze)

Sedative EffectsBalance,

Motor ImparimentAnti-convulsive

Properties

Low et al. (2000)

Anxiolytic Action of Diazepam

Low et al. (2000)

* Affects spatial memory,* Cognitive impairment

Myorelaxation

* AnxiolysisAntidepressionMyorelaxation

Myorelaxation

* Sedation* Anticonvulsant actionAnterograde amnesiaAddiction

GABAA Receptors: Function & Distribution

New Hypnotics• Z-drugs

– Used to treat insomnia, short acting

– Selectively binds to benzodiazepine site on GABAA R with α1 subunit

– Nonbenzodiazepine drugs (Zaleplon (Sonata), Zolpidem (Ambien), Eszopiclone (Lunesta))

– Like benzodiazepines, Zolpidem & Eszopiclone often associated with tolerance & dependence—note that receptors with α1 subunit are associated with addictive behavior

– Potential for abuse

– Some Z drugs have less or no disruption of sleep architecture, less risk of rebound insomnia upon discontinuation (unlike benzodiazepines that actually worsen sleep architecture)

Summary-Anxiolytics, Sedatives, Hypnotics• Benzodiazepines, barbiturates & 5HT1A agonists are anxiolytics

• Benzodiazepines:– increase the affinity of GABA for binding to the GABAA receptor;

– increase frequency of channel opening;

– only effective in the presence of GABA

• GABAA receptor: pentameric

• Agonists bind at interface

• Benzodiazepines bind at interface

• “Knock-in” receptor mutation in transgenic mice allows study of receptor subunits involved in responses to benzodiazepines

• 1 subunit in GABAA receptors mediates benzodiazepine effects of sedation & memory impairment (Z-drugs selective for this type)

• 2 subunit in GABAA receptors mediates benzodiazepine effects of anti-anxiety

• 5 subunit in GABAA receptors – inverse benzodiazepine agonists may enhance cognition

Glycine Receptor: Inhibition in Spinal Reflexes

Glycine Receptors• Pharmacology

• Spinal Reflexes & Inhibitory interneurons– Flexor/Withdrawal reflex– Crossed-Extensor reflex– Stretch reflex– Tendon (Inverse Stretch) reflex– Renshaw inhibition

• Toxins that inhibit neurotransmitter release

HO-CH2

Pharmacology of Glycine Receptors

Competitive antagonist: strychnine

Agonists:• glycine• alanine

Glycine Receptor

• Inhibitory—hyperpolarizes cell membrane• Anion channel• pentameric structure related to GABAA R

Tetanus toxin:inhibits release of glycine in spinal cord.

contract

relax

relax

contract

Crossed Extensor reflex & Flexor/Withdrawal reflex:withdraw from painful stimulus while maintaining balance

Glycine

Painful stimulus:

Stretch Reflex:Balance, walking

Stretch Reflex: maintain balance, walking

Glycine

Stretch Reflex:Balance, homeostasis

Tendon (Inverse Stretch) Reflex:Prevent overstretching of muscle

+ -

Glycine

Spinal Motor Neurons

Renshaw Inhibition:Fine motor control

+

- - -

Glycine & GABA

Syntaxin

SNAP-25

VAMP/Synaptobrevin

Jahn and Scheller (2006)

Purves et al (2007)

SNARE Complex-Vesicle Fusion

Tetanus & Botulinum Toxins: inhibit release of neurotransmitters vesicles

• Produced by anaerobic Clostridium bacteria

• Each toxin is cleaved to produce:– a heavy chain that binds to surface of target nerve cells. Toxin then is

endocytosed, & low endosomal pH aids in translocating light chain across the membrane into the cytosol

– a light chain that is a proteases that cleaves proteins of the SNARE complex involved in vesicle fusion, thereby blocking neurotransmitter release

• Tetanus toxin – taken up by inhibitory interneurons in spinal cord– Cleaves vesicle SNARE synaptobrevin

• Botulinum toxin – taken up by excitatory motor neurons in spinal cord– Types B,D,F&G: cleave vesicle SNARE synaptobrevin– Type C: cleaves plasma membrane SNARE syntaxin– Types A & E: cleaves plasma membrane SNARE SNAP-25

Glycine Receptor• Glycine receptors mediate the major inhibitory

actions in the spinal cord

• Released by inhibitory interneurons involved in spinal reflexes

• Anion channels that allow chloride influx upon glycine binding—hyperpolarizes the membrane potential

• Strychnine is competitive antagonist—causes spinal convulsions

• Tetanus toxin inhibits glycine release in spinal cord—causes muscle rigidity (tetanus)