S.

Urwyler

Neurochemistry Chapter 7: GABA the brake in the brain

Basic Neurochemistry, Chapter 16 (and 5, 37, 55)

s2

-aminobutyric acid (GABA) is the most

important inhibitory neurotransmitter in the brain
O H2N CH2 CH2 C CH2 OH

GABA acts by hyperpolarizing the neuronal membrane through chloride influx via ionotropic (GABA A) receptors. GABAA -60mV
IPSP

-70mV

Slide 2 s2 Whereas excitatory transmitters such as glutamate depolarize the neuronal membrane (i.e. they produce an excitatory postsynaptic potential, EPSP) by increasing Na+ and Ca2+ conductances, GABA hyperpolarizes the membrane by increasing chloride influx, i.e. produces an inhibitory postsynaptic potential. Whether ultimately an action potential is triggered (i.e., whether the threshold potential is reached) depends on the temporal and spatial summation of such excitatory and inhibitory influences. In the spinal cord, glycine plays the same role as GABA in higher parts of the CNS. The glycine receptor also forms a chloride channel. The ionotropic glycine receptor is blocked by strychnine. Left part of the slide from Foster & Kemp Curr. Opinion Pharmacol. 6, 7 (2006)
urwyler, 11/27/2008

GABA- and glutamate metabolism are intricately linked

s1

GABA is made from glutamate by enzymatic decarboxylation

Glutamate decarboxylase uses pyridoxal phosphate (PLP) as a O cofactor:
C O C HO CH2 CH CH2 NH2
O O C CH2 CH2 CH N O O P O O
+

OH

glutamate

Glutamate Decarboxylase (GAD) CO2

HO

C OH

Schiff base intermediate

CH CH2 OH

N H

CH3

O C HO

CH2

CH2

CH2

NH2

-aminobutyric acid (GABA)

Slide 4 s1 Pyridoxalphosphate is a versatile cofactor involved in a wide variety of enzymatic amino acid transformations (transaminations, deaminations, racemizations, decarboxylations). In the case shown here, the bond indicated by the red arrow is labilized in the Schiff base intermediate, leading to decarboxylation and subsequent hydrolysis of the Schiff base. Newly synthesized GABA is taken up into storage vesicles by a transporter which also accepts glycine as a substrate and has also been found in neurons releasing glycine as a transmitter. It could thus be called an inhibitory amino acid transporter.
urwyler, 12/8/2008

s3

The GABA shunt

O H2N CH2 CH2 C CH2 OH

GABATransaminase (GABA-T)
O CH2 O C OH HO C CH2 CH2 O C OH

O C H CH2

Succinic semialdehyde dehydrogenase (SSADH)

Slide 5 s3 The action of synaptically released GABA is terminated by re-uptake into the presynaptic neuron or surrounding glia by plasma membrane GABA transporters. Intracellularly, GABA is then metabolized by the pathways shown here. These reactions, which participate in the synthesis and metabolism of GABA, are called the GABA shunt because the amount of -ketoglutarate that is metabolized via these reactions to succinate is shunted around the succinic dehydrogenase step of the TCA cycle and thus decreases the formation of succinyl-CoA. GABA-T removes GABA by converting it to succinic semialdehyde (SSA) via transamination with -ketoglutarate which re-forms glutamate (red arrows). SSA is oxidized by NAD and SSA dehydrogenase (blue arrows) to NADH+ and succinate, which re-enters the tricarboxylic cycle. Some antiepileptic (anticonvulsant) drugs act by inhibiting GABA reuptake (tiagabine) or the degradation of synaptically released GABA (valproate, which inhibits both GABA-T and SSADH).
urwyler, 12/8/2008

S1

The GABAA receptor belongs to the cys-loop family of ionotropic receptors

Slide 6 S1 LEFT: Schematic of GABAA receptors. (Lower panel) The protein is shown as a pseudosymmetrical membrane-spanning ion channel protein made of five homologous subunits, each of which has four membrane-spanning regions (MSRs, numbered 14) as shown in one of the subunits that is pulled out. (Upper panel) The view from outside the cell (plan view) shows the arrangement of the five subunits (each with four MSRs) around the central core, the chloride ion channel. Also listed are the subunit families that can be utilized in composing each receptor, and the ligands that have binding sites on the receptor. RIGHT: Donut model of GABAA receptor heteropentamer showing ligand binding and subunit interaction domains. The five subunits ( ) and five subunit interfaces (15) are shown, indicating the two different subunit interfaces for each subunit ((- ) and (+)), the ligand binding pockets for GABA and benzodiazepines (BZ), and the various peptide loops (AE) involved in ligand binding and subunit interfaces. These loops are homologous in other members of the ligand-gated ion channel superfamily, including the glycine receptor. The pore in the center represents the chloride channel.
Urwyler, 12/14/2009

S2

Ligands interacting with GABAA receptors

Agonists:
O H2N OH N
N HN O OH

Muscimol

THIP (partial ago)

O H O N
+

Cl CH3

Antagonists:

H O

O O

Bicuculline (competitive)

Picrotoxinin (channel blocker)

Slide 7 S2 Abbreviation: THIP, 4,5,6,7-tetrahydroisoxazolo-pyridin-3-ol Although they are valuable pharmacological tools, compounds acting directly at GABA A receptors are not therapeutically useful. Muscimol (contained in Amanita Muscaria, together with Muscarine!) is an agonist activating the orthosteric GABA recognition site. It has hallucinogenic effects and was therefore used in religious rituals by North American Indians and certain tribes in eastern Siberia. THIP, a conformationally restricted analog of Muscimol, is a partial agonist. The GABA A antagonists Bicuculline and Picrotoxinin are strong convulsant agents.
Urwyler, 12/14/2009

s7

Ligands interacting with GABAA receptors (contd.)

Anesthetic drugs Volatile Intravenous Barbiturates
O

HN

Isoflurane

Halothane

Propofol

N H

Ethanol:

CH3-CH2OH

Pentobarbital (Nembutal)
O

HN

N H

GABAA receptor currents (chloride currents) in Xenopus oocytes. Wallner et al., Proc.Natl.Acad.Sci. USA 100, 15218 (2003)

Phenobarbital

Slide 8 s7 The activity of GABA A receptors is enhanced by a variety of drugs, such as volatile or intravenous anesthetics. Ethanol substantially potentiates receptor activation by GABA already at concentrations easily reached by "social" drinking (the legal blood alcohol concentration limit for driving a car is 11mM [0.05%]). Ethanol easily crosses the blood - brain barrier. 4 and 6 subunit - containing GABA A receptors are particularly sensitive to ethanol. Barbiturates are an important class of sedative / anesthetic, anxiolytic and anticonvulsant [antiepileptic] drugs. They also enhance GABA - mediated chloride currents by enhancing channel opening time. At high concentrations, barbiturates directly activate the GABA receptor. Overdosing of barbiturates can be fatal.
urwyler, 12/8/2008

S3

Benzodiazepines - the success of serendipity

H N N O N

Cl

N O

Cl

Chlordiazepoxide (Librium)
N O

Diazepam (Valium)
O N O

Benzodiazepines are -Anxiolytic -Sedative / hypnotic -Muscle relaxant -Anticonvulsive -and amnesic drugs

N F

Nitrazepam (Mogadon)

Flunitrazepam (Rohypnol)

Slide 9 S3 In the early 1960's the benzodiazepines were introduced as a novel class of anxiolytic / sedative / anticonvulsant and muscle relaxant drugs. They were discovered by chance by Dr. Leo Sternbach at Roche in 1957. (1,4-)Benzodiazepines are considered safe drugs, except in combination with alcohol, due to their pronounced ethanol-potentiating effects. They have some addictive potential, although less than the barbiturates. Benzodiazepines can produce cross-tolerance to ethanol, and they are used in the treatment of alcohol-withdrawal symptoms.
Urwyler, 12/14/2009

s9

Specific binding sites for benzodiazepines were discovered much later than the introduction of these drugs into clinical use
Radioligand binding studies using [3H]diazepam in rat brain membranes

Squires & Braestrup, Nature 266, 732 (1977) Mhler & Okada, Science 198, 849 (1977)

Slide 10 s9 The existence of specific binding sites for benzodiazepines in the brain was discovered independently by two groups at the same time. Both used radioligand binding techniques to demonstrate specific binding of [3H]diazepam to brain membranes. Left: saturation curve; circles: specific binding, triangles: non-specific binding. Inset: linearized transformation of the saturation curve ("Scatchard plot"). Right: Top: stereospecific displacement of [3H]diazepam by two other benzodiazepines. Bottom: the potencies of benzodiazepines as displacers of [3H]diazepam correlates with their potencies as muscle relaxant agents in vivo.
urwyler, 12/8/2008

s10

Benzodiazepines are allosteric modulators of GABAA receptors

+ CDPX (100M) control

Kelly et al., Br.J.Pharmacol. 135, 248 (2002) (chloride currents in Xenopus oocytes)

Choi et al., J.Neurophysiol. 45, 621 (1981) (GABA effects in chick sensory neurons)

Slide 11 s10 Benzodiazepines are positive GABA A receptor modulators. Left: enhancement of GABA - mediated chloride currents in Xenopus oocytes. Right: Chlordiazepoxide enhances the potency of GABA in chick sensory neurons (shift of the concentration-response curve without a change in the maximal GABA effect). Radioligand binding experiments have also demonstrated the mutual enhancement of binding affinities between GABA and benzodiazepines. In contrast to the directly acting agonist muscimol, benzodiazepines do not have prohibitive side effects like hallucinations. Positive allosteric modulators act more physiologically, because they are effective only when and where the receptor is activated by endogenous GABA. An agonist, on the other hand, activates every receptor with which it comes into contact, independently of synaptic signaling.
urwyler, 12/8/2008

S4

Benzodiazepines have a wide range of intrinsic efficacies

O N
N N O C

O N
O

C O

Cl

N
F

N O

S O

Diazepam (agonist)

Flumazenil (antagonist)
O

Ro19-4603 (inverse agonist)

Intrinsic efficacy
N N C O H N Br O

0
N N

Intrinsic efficacy
O C O

N3 O

Ro15-4513 (partial inverse agonist)

Bretazenil (partial agonist)

Slide 12 S4 Partial or full benzodiazepine agonists enhance the effects of GABA. Flumazenil binds to the benzodiazepine site as a neutral antagonist. Partial or full inverse agonists at the benzodiazepine site diminish the effects of GABA. Note that the terms "agonist" or "antagonist" are referring to the actions of these compounds at the benzodiazepine site. This means that a "benzodiazepine agonist" enhances the effects of GABA, but it is not an agonist at the GABA receptor, because it does not activate it directly. Similarly, a "benzodiazepine antagonist" like flumazenil does not antagonize the effects of GABA, but it blocks the enhancing or diminishing effects of benzodiazepine agonists or inverse agonists, respectively. Flumazenil is clinically used to reverse sedation due to a benzodiazepine overdose, or to manage the anesthetic effects of benzodiazepines.
Urwyler, 12/14/2009

S5

A conserved histidine residue is essential for diazepam sensitivity of GABAA receptors

Rudolph et al., Nature 401, 796 (1999)

Chloride currents in HEK-293 cells recombinantly expressing GABAA receptors ( Benson et al., FEBS Lett. 431, 400 [1998])

Slide 13 S5 1-, 2-, 3- and 5-subunit - containing GABA A receptors are diazepam - sensitive. They all have a conserved histidine residue at a homologous position. Diazepam-insensitive 4- and 6-subunits have an arginine at the corresponding positions. Replacing the essential histidine residue by an arginine by site-directed mutagenesis abolishes the diazepam-sensitivity of 1-, 2-, 3and 5-subunit - containing GABA A receptors. Top left: Autoradiography in rat brain using the benzodiazepine radioligand [3H]Ro15-4513 (see previous slide). It is displaceable by diazepam in the wild-type mouse, but not in a 1 H101R - mutant. The binding of the radioligand itself is apparently not affected by the mutation. Bottom: chloride currents are enhanced by diazepam in a recombinant cell line expressing wild-type 2-subunit containing receptors, but not in a 2 H101R - mutant.
Urwyler, 12/14/2009

s13

Making the light choice...

Slide 14 s13 Mice have an innate fear of light. In a two-compartment cage, they will prefer the dark compartment over the light one. Under the influence of an anxiolytic drug, however, the time they spend in the lit compartment will increase. In genetically engineered mice carrying the alpha2 H101R mutation, diazepam will not increase time spent in the lit compartment (next slide).
urwyler, 12/8/2008

s14

Anxiolytic, but not sedative and amnesic properties of diazepam are mediated by 2 containing GABAA receptors

Loew et al., Science 290, 131 (2000)

Slide 15 s14 In the light/dark box test, diazepam increases time spent in the lit area in wild-type, but not in 2 H101R mutant mice. This difference is not produced by the corresponding 3-mutation. On the other hand, sedation (decrease in locomotor activity) produced by diazepam is not affected by this 2 subunit - mutation. Likewise, motor impairment, assessed by the loss of ability to stay on a rotating rod, is unchanged in the "knock-in" mice carrying the 2 H101R - mutation.
urwyler, 12/9/2008

s15

Subtype-selective benzodiazepines: drugs of the future?

Sedation, Anterograde amnesia

Anxiolysis, myorelaxation

2-subtype - selective benzodiazepines might be novel anxiolytic drugs without the amnesic and sedative side effects !
Mhler et al., J.Pharmacol.Exp.Ther. 300, 2 (2002)

Slide 16 s15 Experiments similar to the ones shown in the previous slide have demonstrated that the sedative and amnesic, but not the anxiolytic properties of diazepam are mediated by 1 containing GABA A receptors Rudolph et al., Nature 401, 796 [1999]). 2 - subunits are present in only about 15% of the total diazepam-sensitive GABA A receptors. Targeting this small subpopulation should allow to find novel anxiolytic drugs with less side effects.
urwyler, 12/9/2008

s16

Neuroactive steroids are endogenous enhancers of GABAA receptor function

Progesterone

Allopregnanolone

Reddy, Trends Pharmacol. Sci. 24, 103 (2003)

Slide 17 s16 Endogenous neurosteroids are formed in two enzymatic reductive steps from steroid hormone precursors. They can be synthesized locally in neurons. At low nanomolar concentrations, observed during stress or alcohol intoxication, they potentiate GABA currents, whereas at higher (low micromolar) concentrations, which occur e.g. during childbirth, they directly activate the receptor. Although the effects of neurosteroids resemble those of barbiturates and benzodiazepines, they clearly act via a separate binding site on the receptor complex. Allopregnanolone is derived from progesterone and therefore may play a role during the menstrual cycle and pregnancy. On the other hand, THDOC is made from deoxycorticosterone, the levels of which are elevated in response to the hypophyseal-pituitary axis (HPA) in stressful situations. Although the physiological significance of neurosteroids need further clarification, it has been suggested that neurosteroids play an important role in human stress-sensitive conditions such as epilepsy (to reduce seizure-susceptibility!), post-traumatic stress disorder and depression. They might therefore have a therapeutic utility in stress-sensitive neurological and psychiatric conditions.
urwyler, 12/9/2008

The GABA-analog baclofen helps in the discovery of a novel receptor

O H2N OH

- Early 1970s: introduction of p-chloro-phenylGABA in clinical use as an antispastic agent. - 1980/81: The pharmacological actions of baclofen in vitro are not bicuculline-sensitive. Specific binding sites for baclofen demonstrated (Bowery et al., Nature 283, 92 [1980], Hill & Bowery, Nature 290, 149 [1981]). Postulation of the existence of an additional receptor for GABA, termed GABAB. - 1997: Molecular cloning of the elusive GABAB receptor (Kaupmann et al. Nature 386, 239 [1997])

Cl

(Baclofen, Lioresal)

s17

G protein - coupled GABAB receptors are a heteromeric assembly of two different subunits

Couve et al., Mol.Cell.Neurosci. 16, 296312 (2000)

Slide 19 s17 - The GABAB receptor consists of two related subunits, which are separate gene products. Both belong to family C GPCRs. - The extracellular domain lacks the cysteine-rich region found e.g. in mGluRs - No actual GABAB receptor subtypes exist, but splice variants have been identified (e.g. in the extracellular "Sushi domains" Su1 and Su2). - Only GABAB1 binds agonists and competitive antagonists - GABAB2 couples to the G-protein and conveys the intracellular signal. It allosterically interacts with GABAB1 and enhances agonist potency. Moreover, it is essential for the cell surface expression of the receptor complex. GABAB1 monomers are retained in the endoplasmic reticulum and not transported to the plasma membrane.
urwyler, 12/15/2008

s20

Pre-and postsynaptic effects of GABAB receptors

GABA BR

Presynaptic autoreceptors inhibit the release of GABA (feedback mechanism) Presynaptic heteroreceptors inhibit the release of other neurotransmitters: glutamate dopamine norepinephrine neuropeptides

Cl

K+

GABA AR GABAA
early IPSP

+ CGP55845 3M control
GABAB
late IPSP

Slide 20 s20 Postsynaptically, GABA B receptor activation results in a prolongation of the inhibitory postsynaptic potential (IPSP) by activation of inwardly rectifying potassium channels. Addition of a GABA B receptor antagonist (e.g. CGP55845) eliminates this slow, long lasting component of the IPSP. Presynaptically, GABA B receptor activation inhibits voltage-sensitive calcium channels and thereby the release of GABA (autoreceptors) and other neurotransmitters (heteroreceptors).
urwyler, 12/15/2008

s21

Essential amino acids for GABA binding are not conserved in GABAB2

Kniazeff et al., J.Neurosci. 22, 7352 (2002)

Slide 21 s21 Ser246, Glu465 and Tyr366 are not conserved in GABA B2. Therefore, this subunit has lost the ability to bind GABA. The lack of evolutionary pressure on GABA B2 is illustrated by the phylogenetic relationships shown to the left. When compared to GABA B1, it is even more striking when only the actual binding region (b), instead of the whole venus flytrap module (a), is taken into consideration.
urwyler, 12/16/2008

s22

Ligands interacting with GABAB receptors

Agonists: Agonists:
H2N C
H

O OH O
H2N

Allosteric modulators:
OH

GABA
HO C

OH
N
HO

S N N H O

GHB
O H2N P OH H

OH
Cl

N H N O

APPA

(R)-(-)-baclofen

CGP7930

GS39783

Antagonists:
O H2N O P OH O
O OH N H OH
Cl OH CH3

O P OH

Cl OH H N O P

CGP35348

CGP56999

CGP55845

Slide 22 s22 GHB (gamma-hydroxybutyrate) has anesthetic / hypnotic actions and a pharmacological profile which is somewhat different from that of baclofen. In fact, additional specific GHB binding sites have been identified, the exact function of which is not clear yet. (R)-(-)baclofen is the active enantiomer. However, the racemic form of baclofen is on the market as an antispastic agent. To date, baclofen is the only marketed drug targeting the GABA B receptor. APPA: 3-aminopropylphosphinic acid
urwyler, 12/15/2008

S6

Effects of agonists and positive allosteric modulators on GABAB receptors in vitro:

Radioligand binding:
% specific binding
100 75 50 25

GABA Baclofen APPA

GTP()35S binding:
CGP7930: cpm above basal activity
7500

30M 10M
5000

controls + 30M GS39783

3M 1M 0 (control)

0 -10

-9

-8

-7

-6

-5

-4

-3

2500

log [displacer] (M)

0 -8 -7 -6 -5 -4

log [GABA] (M)

Urwyler et al., Mol.Pharmacol. 60, 963 (2001) Urwyler et al., J.Pharmacol.Exp.Ther. 307, 322 (2003)

Potassium currents in Xenopus oocytes:

Slide 23 S6 Panel A: Stimulation of GTP()35S binding in membranes from a chinese hamster ovary (CHO) cell line recombinantly expressing GABAB receptors. CGP7930 not only increases the potency, but also the maximal efficacy of GABA. Panel B: GS39783 enhances agonist binding affinities (displacement of the antagonist radioligand [3H]CGP62349 from GABAB receptors in rat cortical membranes). Note that the curves are slightly biphasic (two affinity states, coupled and not coupled to the G-protein). Panel C: Potassium currents in Xenopus oocytes transfected with the two GABA B subunits and inwardly rectifying potassium channels. Two first peaks: presence of a saturating GABA concentration. Peaks 3 and 4: A low GABA concentration with and without CGP7930. Note that the allosteric modulator alone, applied before GABA, has no effect.
Urwyler, 12/14/2009

GABAB receptor stimulation inhibits adenylyl cyclase activity in vitro....

Inhibition of forskolin-stimulated cAMP formation by GABA in a recombinant cell line expressing GABAB receptors:

cAMP (pmoles / well)

+ forskolin
10.0 7.5 5.0 2.5

control +GS39783, 10M

basal
0.0 -9 -8 -7 -6 -5

log[GABA] (M)
Urwyler et al. Neuropharmacology 48, 343 (2005)

.... and in vivo.....

s18

In vivo microdialysis: a powerful biochemical sampling technique

Bourne, Clin.Exp.Pharmacol.Physiol. 30, 1624 (2003)

Slide 25 s18 Left: Experimental set-up for the recovery of endogenous extracellular (interstitial) substances in an awake animal, showing (a) the microfraction collector (b) the microsyringe / perfusion pump (c) liquid swivel crane (this counterbalanced apparatus allows the animal free/unrestricted movement while allowing the collection of perfusate) and (d) the observation cage. Right: Diagrammatic representation of passive transfer of endogenous substances into the microdialysis probe after their release into the synaptic cleft and from the extracellular environment. The dialysate flows down the inner of the two concentric tubes of the microdialysis probe and exits via a small hole at the bottom of the tube. It then flows upwards, past the osmotic membrane, capturing the substances that have diffused across the membrane, such as neurotransmitters and other low molecular weight substances. Other (e.g. U-shaped) types of microdialysis probes can also be used. The microdialysis probe not only serves to collect substances from brain tissue, but can also be used to locally apply drugs.
urwyler, 12/15/2008

s19

GABAB receptor-mediated inhibition of cyclic AMP formation in rat striatum in vivo A A

B C
Gjoni et al., J.Neurochem. 96, 1416 (2006)

Slide 26 s19 Measurement of the effects of baclofen and GS39783 on cAMP formation in rat striatum in vivo by microdialysis. The production of cAMP was stimulated twice by applications of a water-soluble forskolin analog through the dialysis probe. S1 served as the control stimulus. When test compounds were applied, they were given before S2. The normalization to S1 (S2/S1-ratios calculated from areas under the peaks) allows each animal to be its own control. The phosphodiesterase inhibitor IBMX was present in the perfusate throughout. Panel A: Traces of extracellular cAMP efflux. Solid line: control (S2/S1 ratio: 1.0 0.05). Dotted line: effect of 500 M R()-baclofen (S2/S1 ratio: 0.61 0.03). A water-soluble forskolin-analog was perfused through the microdialysis probe twice for 10 min (S1 and S2, black bar) and R()-baclofen for 20 min before and 10 min during S2 (grey bar). To account for differences in basal values among animals, extracellular cAMP levels are expressed as a percentage of baseline. All data shown are mean values with their standard errors. Panel B: Concentrationresponse curve showing effects of R()-baclofen. The inset shows the effect of the GABAB antagonist CGP56999. CGP56999 (30 M) was coinfused with R()-baclofen (200 M) for 30 min before and during S2. Black bar, control; striped bar, baclofen alone; checkered bar, baclofen + CGP56999. Panel C: Effects of GS39783 (10, 30 and 100 mg/kg) combined with 1 M R()-baclofen and antagonization with 10 M CGP56999 (both applied through the microdialysis probe). GS39783 was given orally 1h before S2.
urwyler, 12/15/2008

Summary: GABAergic inhibition

Bettler & Tiao, Pharmacol. Ther. 110, 533 (2006)