Ž . Brain Research Reviews 29 1999 196–217 Full-length review An update on GABA receptors A Ashok K. Mehta, Maharaj K. Ticku ) Department of Pharmacology, The UniÕersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl DriÕe, San Antonio, TX 78284-7764, USA Accepted 20 October 1998 Abstract Recent advances in molecular biology and complementary information derived from neuropharmacology, biochemistry and behavior have dramatically increased our understanding of various aspects of GABA receptors. These studies have revealed that the GABA A A Ž receptor is derived from various subunits such as a – a , b – b , g – g , d, ´, p, and r . Furthermore, two additional subunits b , 1 6 1 3 1 3 1–3 4 . Ž . g of GABA receptors in chick brain, and five isoforms of the r-subunit in the retina of white perch Roccus americana have been 4 A identified. Various techniques such as mutation, gene knockout and inhibition of GABA receptor subunits by antisense oligodeoxynu- A cleotides have been used to establish the physiologicalrpharmacological significance of the GABA receptor subunits and their native A receptor assemblies in vivo. Radioligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinity chromatography and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of native GABA receptor assemblies. Partial agonists of GABA receptors are being developed as anxiolytics which have fewer and less severe A A side effects as compared to conventional benzodiazepines because of their lower efficacy and better selectivity for the GABA receptor A subtypes. The subunit requirement of various drugs such as anxiolytics, anticonvulsants, general anesthetics, barbiturates, ethanol and neurosteroids, which are known to elicit at least some of their pharmacological effects via the GABA receptors, have been investigated A during the last few years so as to understand their exact mechanism of action. Furthermore, the molecular determinants of clinically important drug-targets have been investigated. These aspects of GABA receptors have been discussed in detail in this review article. A q 1999 Elsevier Science B.V. All rights reserved. Keywords: GABA receptors; Anxiolytics; Anticonvulsants; General anesthetics; Ethanol; Neurosteroids A Contents 1. Introduction ........................................................................ 197 2. GABA receptor subunits ................................................................ A 197 3. Subunit composition of native GABA receptors and their binding characteristics .................................. A 198 4. Molecular determinants of GABA receptors ...................................................... A 200 5. Physiologicalrpharmacological role of various GABA receptor subunits and their receptor assemblies ....................... A 201 6. Interaction of drugs with GABA receptors ....................................................... A 203 6.1 Anxiolytics ...................................................................... 203 6.2 Anticonvulsants ................................................................... 203 6.3 General anesthetics .................................................................. 204 6.4 Barbiturates ...................................................................... 204 6.5 Ethanol ........................................................................ 205 6.6 Neurosteroids ..................................................................... 206 6.7 Miscellaneous .................................................................... 206 ) Corresponding author. Fax: q1-210-567-4226; E-mail: [email protected]0165-0173r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0165-0173 98 00052-6
Transcript
Ž .Brain Research Reviews 29 1999 196–217
Full-length review
An update on GABA receptorsA
Ashok K. Mehta, Maharaj K. Ticku )
Department of Pharmacology, The UniÕersity of Texas Health Science Center at San Antonio, 7703 Floyd Curl DriÕe, San Antonio, TX 78284-7764, USA
Accepted 20 October 1998
Abstract
Recent advances in molecular biology and complementary information derived from neuropharmacology, biochemistry and behaviorhave dramatically increased our understanding of various aspects of GABA receptors. These studies have revealed that the GABAA A
Žreceptor is derived from various subunits such as a –a , b –b , g –g , d, ´, p, and r . Furthermore, two additional subunits b ,1 6 1 3 1 3 1– 3 4. Ž .g of GABA receptors in chick brain, and five isoforms of the r-subunit in the retina of white perch Roccus americana have been4 A
identified. Various techniques such as mutation, gene knockout and inhibition of GABA receptor subunits by antisense oligodeoxynu-A
cleotides have been used to establish the physiologicalrpharmacological significance of the GABA receptor subunits and their nativeA
receptor assemblies in vivo. Radioligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinitychromatography and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of nativeGABA receptor assemblies. Partial agonists of GABA receptors are being developed as anxiolytics which have fewer and less severeA A
side effects as compared to conventional benzodiazepines because of their lower efficacy and better selectivity for the GABA receptorA
subtypes. The subunit requirement of various drugs such as anxiolytics, anticonvulsants, general anesthetics, barbiturates, ethanol andneurosteroids, which are known to elicit at least some of their pharmacological effects via the GABA receptors, have been investigatedA
during the last few years so as to understand their exact mechanism of action. Furthermore, the molecular determinants of clinicallyimportant drug-targets have been investigated. These aspects of GABA receptors have been discussed in detail in this review article.A
q 1999 Elsevier Science B.V. All rights reserved.
Keywords: GABA receptors; Anxiolytics; Anticonvulsants; General anesthetics; Ethanol; NeurosteroidsA
Ž .g-Aminobutyric acid GABA is a major inhibitoryneurotransmitter in the vertebrate central nervous system.GABA activates three different receptor classes such asGABA , GABA and GABA receptors. GABA recep-A B C A
w xtors are ligand-gated chloride ion channels 21,286 . Thesereceptors are activated by GABA, muscimol and isogu-
Žvacine, and are inhibited by bicuculline, gabazine SR. Ž . w x95531 and q -b-hydrastine 345 . GABA receptors areB
Ž . Ž . Žactivated by GABA, y -baclofen, " -4-amino-3- 5-.chloro-2-thienyl butanoic acid and 3-aminopropyl-
Ž . Ž .methyl phosphinic acid SKF 97541 , and are inhibited byw xphaclofen, saclofen and 2-hydroxysaclofen 275 . These
receptors are known to be coupled to Ca2q or Kq channelsvia G proteins so as to activate the second messenger
w xsystems within the cell 21,24 . GABA receptors areC
derived from various isoforms of the r-subunit, and aredirectly associated with chloride ion channels. These re-ceptors are activated by GABA and certain conformation-ally restricted analogues of GABA such as cis-4-
Ž .aminocrotonic acid CACA and trans-4-aminocrotonicŽ .acid TACA , and are inhibited by imidazole-4-acetic acid
w x wŽ .22 and 1,2,5,6-tetrahydropyridin-4-yl methylphosphinicx Ž . w xacid TPMPA 260 but are insensitive to bicuculline,
w xbarbiturates, benzodiazepines and baclofen 83,253,353 .Recently, it has been proposed that GABA receptorsC
should be classified as a specialized set of the GABAAw xreceptors 10 . The GABA receptors are of great impor-A
tance as they play a pivotal role in the regulation of brainexcitability, and many important drugs such as benzodi-azepines, barbiturates, neurosteroids, ethanol, and some ofthe anticonvulsants and general anesthetics interact withthese receptors so as to elicit their pharmacological effects.
2. GABA receptor subunitsA
GABA receptor is a transmembrane hetero-oligomericA
protein which is expressed in the peripheral and centralnervous system. The deduced amino acid sequences ofGABA receptor subunits show significant sequence iden-A
Ž .tity 20% to 30% with other ligand-gated ion channelssuch as the nicotinic acetylcholine receptor, glycine recep-tor and 5-HT receptor. Several subunit classes and iso-3
forms within each class of the GABA receptor have beenA
cloned in the mammalian brain. Amino acid identity amongisoforms of the same class is ;70% whereas the identityamong classes is ;30%. Various subunits of the GABA
receptors are a –a , b –b , g –g , d, ´, p, and r –r1 6 1 3 1 3 1 3w x34,55–57,61,121,177,181,230,232,280,365 . Receptor as-semblies derived from various isoforms of the r-subunitare suggested to be classified as GABA receptorsCw x230,280 as these receptors are insensitive to both bicu-culline and baclofen. However, it has been proposed re-cently that the r-subunit containing receptors are best
w xclassified as a specialized set of the GABA receptors 10A
since r-subunits are structurally part of the family ofGABA receptor subunits despite of their distinctive regu-A
w xlatory binding sites 230,280 . Furthermore, two additionalŽ .subunits b , g of GABA receptors in chick brain4 4 A
w x12,119 , and five isoforms of the r-subunit in the retina ofŽ . w xwhite perch Roccus americana 254 have been identi-
fied. There are also reports that some of the subunits existw xas splice variants 12,118,156,158,346 . The g -subunit is2Ž . Ž .expressed as g short form and g long form vari-2S 2L
ants. The g variant contains an eight amino acid insert2L
between TM3 and TM4 which provides a potential phos-w xphorylation site 156,346 . Two splice variants of the
w xa -subunit have also been reported 158 . However, the6
short form of a -subunit does not form functional recep-6w xtors 158 . Other examples of subunit splice variants are
w xchicken b and b genes 12,118 , and human b gene2 4 3w x154 . Distribution of major subunits in various regions of
w xbrain has been investigated 354 . Briefly, the a -subunit1
is present predominantly in the cerebellum, whereas thehippocampus has low levels. In contrast, the a -subunits2 – 5
are present predominantly in the hippocampus, whereascerebellum is practically devoid of these subunits. Cerebralcortex has intermediate levels of a -subunits and low1 – 4
levels of a -subunit. The a -subunit is present in the5 6
cerebellar granule cells, whereas hippocampus and cerebralcortex are devoid of this subunit. The relative distributionof b - and b -subunits is as follow: hippocampus)cortex1 3
)cerebellum, whereas the rank for the b -subunit levels is2w xcerebellum ) cortex ) hippocampus 171,354 . Interest-
ingly, the g -subunit mRNA is expressed predominantly in1
amygdala and septum, in contrast to most of the GABAA
receptor subunits which are mainly expressed in the cere-bral cortex and cerebellum, and shows little change with
w xpostnatal development 343,354,359 . The g -subunit is2
present in abundant amount in almost every region of thebrain, whereas the g -subunit is present mainly in the3
w xcortex and basal nuclei 354 . There is also report that thelevels of g -subunit are more abundant than the g -sub-2S 2L
unit levels in the hippocampus, cerebral cortex and olfac-w xtory bulb 103 . In contrast, the inferior colliculus, medulla
( )A.K. Mehta, M.K. TickurBrain Research ReÕiews 29 1999 196–217198
w xand cerebellum have more g - vs. g -subunit 103 . The2L 2S
d-subunit is present predominantly in cerebellum, whereashippocampus and cortex have low levels of this subunitw x354 . Thalamus contains intermediate levels of d-subunit,
w xand high levels of a -subunit 354 . The ´-subunit mRNA4
is present in amygdala, thalamus and subthalamic nucleusw x57 . On the other hand, the p-subunit has been reported to
w xbe present in lung, thymus, prostate and uterus 121 .Although, various isoforms of the r-subunit are known to
w xbe localized in the retina 55,56,73,195,230,254 , it hasbeen shown recently that the r -subunits are also ex-1 – 3
pressed in the rat brain regions such as superior colliculus,w xcerebellar Purkinje cells and hippocampus 23 . Im-
munoblot studies using antibodies to various subunits ofGABA receptors have revealed the molecular mass ofA
these subunits as follows: a s50–51 kDa, a s52–531 2
kDa, a s58–61 kDa, a s66–67 kDa, a s53–55 kDa,3 4 5
a s57–58 kDa, b s57kDa, b s54–57 kDa, b s576 1 2 3
kDa, g s45–51 kDa, g s43–49 kDa, g s45 kDa,1 2 2Swg s47 kDa, g s43–46 kDa, and ds54 kDa 16–2L 3
19,61,69,72,149,150,152,153,171,178,194,195,207,257,x259,298,305 . The polyclonal anti-a antibody, raised by1
us in a rabbit against the synthetic peptide correspondingto the amino acids 1–15 of the rat a -subunit of GABA1 A
receptors, recognized a protein band of 51 kDa in im-munoblots, whereas the polyclonal anti-b antibody, raised2
by us in a rabbit against the synthetic peptide AGLPRHSF-w xGRNA 171 corresponding to the amino acid residues
382–393 of the rat b -subunit of GABA receptors, rec-2 A
ognized a protein band of 55 kDa in immunoblots.
3. Subunit composition of native GABA receptors andA
their binding characteristics
GABA receptor is a pentameric assembly derivedA
from a combination of various subunits. Elucidation ofthese assemblies of native GABA receptors is a veryA
challenging task since several different types of receptorassemblies can be derived theoretically from a combina-tion of various subunits. There are reports indicating thatthe a , b , and g -subunits co-exist in many native1 2r3 2
w xGABA receptors 16,149,298 . Furthermore, it is knownAwthat two different isoforms of the a- 5,8,19,69,72,
x w x151,152,160,176,207,242,243,330 , b- 171 , or g-subunitw x17,150,151,257 can co-localize in the same receptor as-sembly. However, the preferred pentameric combination
w xincludes two a , two g, and one b-subunits 8 , or two a ,w xtwo b, and one g-subunits 39 . Recently, it has been
suggested that a total of four alternating a and b-subunitsare connected by a g-subunit in a pentameric GABAA
w xreceptor assembly 319 . The preferred stoichiometry ofGABA receptor subtype probably varies among differentA
brain regions. In an attempt to elucidate the completepentameric composition of a brain receptor, the cerebellarreceptor assemblies have been proposed as a a g -1 6 2S
g b , a a b g d, a b g , a b g , a a -2L 2r3 1 6 2r3 2 1 2r3 2 6 2r3 2 1 6wb g , a b g d , and a b d 36,37,151,2r 3 2 1 2r 3 2 6 2r 3
x152,159,160,207,243,258 . However, recent studies havedemonstrated that d-subunit does not co-localize with g-
w xsubunit in native GABA receptor assemblies 4,259 . TheA
diazepam-insensitive benzodiazepine-site in the humancortex has been reported to be composed of the a -, b -,4 2
w xand g -subunits 357 .2L
Although co-expression of the a-, b- and g-subunits isrequired for the formation of fully functional GABAA
receptors and most of the native GABA receptor assem-A
blies consist of these subunits yet the co-localization ofthese three types of subunits is not an absolute requirementfor the formation of the GABA receptor. It is known thatA
the mice devoid of the g -subunit were able to express2
functional GABA receptors, though devoid of the benzo-A
diazepine binding site, in unaltered numbers from thew x w xremaining subunits 101 . In contrast, when the a - 88 ,5
w x w xa - 127,140 , or b -subunit 126 gene was inactivated,6 3
some of the brain GABA receptors were lost. TheseA
observations suggest that the a- and b-subunits, unlike theg -subunit, are essential components for the receptor as-2
sembly and membrane targeting of the GABA receptors.A
Furthermore, it has been suggested that each receptorsubtype has its own target identity depending on thesubunits, and highly selective recognition processes oper-ate to ensure differential assembly and subcellular distribu-
w xtion of the receptor subtypes within a cell 88–90,216,228 .During the last few years, localization of various bind-
ing sites on the GABA receptors has been investigated.A
These studies have indicated that the high affinityGABArmuscimol binding site is localized at the interface
w xof the a- and b-subunits of GABA receptors 246,364 .A
The b-subunit of GABA receptors has been suggested asA
a necessary requirement for the TBPS binding siteŽ . w xpicrotoxin site 288,364 . Although the homo-oligomericGABA receptors derived from b -subunit exhibit a spe-A 3
w35 xcific high affinity binding for S TBPS, the GABAA
receptor assemblies consisting of a b g - or a b -sub-1 3 2 1 3w35 xunits have higher affinity for S TBPS over those consist-
w xing of b g - or b -subunits 288,364 . Benzodiazepine3 2 3Ž .BZ site is localized at the interface of the a- and
w xg-subunits of GABA receptors 355,364 . Therefore, theA
GABA receptor assemblies containing two isoforms ofA
the a- as well as g-subunit would have two benzodi-w xazepine sites in the same receptor assembly 152 . Recep-
tors containing the a -subunit mimic BZ I pharmacology,1
whereas those containing the a -, a -, or a -subunit2 3 5wvariant display BZ II pharmacology 16,189,194,207,
x247,363 . BZ II receptors have lower affinity for CL218872, b-CCM and zolpidem as compared to BZ I recep-
w xtors 16,189,194,207,247,363 . However, receptors con-taining the a -subunit variant lack affinity for zolpidem5
w xand its analogues 109,194,197,207,247 but have highŽ . w xaffinity for Ro 15-4513 sarmazenil 109,197 , a partial
inverse agonist of the benzodiazepine site. BZ I receptors,
GABA receptor assemblies containing a - and g-sub-A 1
units, are present predominantly in the cerebellum but arescarce in the hippocampus, whereas BZ II receptors areexpressed in abundance in the hippocampus and almost
w xabsent in the cerebellum 80,81,197,231 . However, bothreceptor types, i.e., BZ I and BZ II receptors are equally
w xexpressed in the cerebral cortex 81,231 . A specific lig-w3 x Ž .and, H zolpidem K f7 nM , for the BZ I receptors hasd
w x w3 x Ž .been developed 6 . Similarly, H L-655,708 K f2 nMdw x w3 x Ž . w x256 and H RY 80 K f0.7 nM 287 have beend
developed as ligands for BZ II receptors derived from thea -subunit. Conventional benzodiazepines such as di-5
azepam, flunitrazepam and clonazepam failed to bind tothe GABA receptor assemblies containing benzodi-A
azepine site derived from the a - or a -subunit, whereas4 6
Ro 15-4513 and some of the benzodiazepine receptorpartial agonists have high affinity for these diazepam-in-
w xsensitive receptors 152,153,178,198,258,313 . Moreover,inverse agonists such as DMCM, b-CCE, b-CCM, andFG-7142 showed 7 to 10 times higher affinity for
w xthe a b g vs. a b g GABA receptors 357 .4 2 2L 6 2 2L Aw3 x w xH Muscimol binds with high affinity to the a - 153,3524
w xas well as a -subunit 152 containing GABA receptors.6 A
The a -subunit containing receptor assemblies are local-6w xized in cerebellar granule cells 178,258,305 , whereas
those containing the a -subunit variant are localized in4
various brain regions such as cerebral cortex, hippocam-pus, thalamusrhypothalamus, striatum and olfactory bulbw x w x19,153,352 , and retina 195 . Radioligand binding to theimmunoprecipitated receptors using polyclonal antibodiesto various g-subunits has revealed that the g - or g -sub-1 3
unit containing receptor assemblies are of low abundancew x17 . Furthermore, the g -receptor population displayed a3
reduced affinity for clonazepam and flunitrazepam, andw xvirtually lacked sensitivity to zolpidem 17 . The g -recep-1
tor assembly displayed low affinity for most of the benzo-diazepine site ligands except flunitrazepam, and can bedifferentiated from those containing the g - or g -subunit2 3
by its low affinity for the inverse agonist b-CCM and itslack of affinity for the partial inverse agonist Ro 15-4513Ž . Ž .sarmazenil as well as for the Ro 15-1788 flumazenil , a
w xcompetitive antagonist of the benzodiazepine site 17 . It isthus evident that the g -subunit receptor assemblies are not1
involved in mediating all major effects of benzodiazepinesin vivo since these effects are susceptible to blockade byflumazenil. Replacement of the g-subunit by a d-subunit inthe expression studies produces a GABA receptor whichA
w xis not potentiated by benzodiazepines 269,270,281 . Poly-clonal antisera raised against the d-subunit has been re-
w3 xported to immunoprecipitate the H Ro 15-1788 bindingw xsites 18 , thereby suggesting that the GABA receptorsA
containing d-subunit have a benzodiazepine binding sitew xwhich has a novel pharmacology 207 . Although the
d-subunit is reported to co-localize with a -, a -, a -,1 3 5
a -, and b -subunits in various receptor assemblies6 2r3w x140,207 , it does not co-localize with the g-subunit in
native GABA receptors according to recent studiesAw x4,259 . These results make it difficult to explain thediscrepancy between the expression study data and theimmunoprecipitation results. However, recent studies haveindicated that the polyclonal antiserum raised against d-
w3 xsubunit immunoprecipitates only the H muscimol bindingw3 x w3 xsites, whereas the H Ro 15-1788 or H flunitrazepam
binding sites are not immunoprecipitated by this antibodyw xin the rat brain 4,259 in contradiction to the findings of
w xBenke et al. 18 . Transfection of cells with a combinationof a-, b- and ´-subunits results in the expression of
w35 x w xGABA-activated currents and S TBPS binding sites 57 .However, these transfected cell membranes did not bind
w3 x w3 x w xeither H Ro 15-1788 or H flunitrazepam 57 . Transfec-tion of cells with a combination of a , b, and p-subunits
w3 xresults in the expression of both H muscimol andw35 x w3 xS TBPS binding sites but no H Ro 15-1788 orw3 x w xH flunitrazepam binding sites were detected 121 . Whenthe p-subunit was expressed with arbrg-subunit combi-nation in transfection system, there was a reduction in the
w3 x w xdensity of H Ro 15-1788 binding sites 121 . In contrastto earlier view regarding the homomeric nature of ther-subunit receptor assemblies, it has been shown recentlythat the rat retinal receptor assemblies derived from thesesubunits are heteromeric and composed of at least r - and1
w xr -subunits 365 , and both the r - and r -subunits are2 1 2w xnecessary to form a functional receptor assembly 23 .
Radioligand binding to the immunoprecipitated recep-tors using subunit-specific antibodies has been used as atool to establish and understand the composition and phar-macology of native GABA receptor assemblies. TheseA
studies have been conducted primarily in cerebral cortex,cerebellum and whole brain using ligands of the GABA
Žw3 x .binding site H muscimol and the benzodiazepine siteŽw3 x w3 x w3 x .H flunitrazepam, H Ro 15-1788 and H Ro 15-4513 .The reported percentage of the binding sites immunopre-cipitated by antisera to various subunits of GABA recep-A
tors in the adult brain regions are as follows: a s70–97%,1
a s4–28%, a s12–24%, a s0–15%, a s4–14%,2 3 4 5Ž .a s30–39% cerebellum , b s2–32%, b s55–96%,6 1 2
b s 19–52%, g s 0–19%, g s 50–94%, g s 31–3 1 2 2S
52%, g s 37–65%, g s 0–18%, and d s 0–23%2L 3w x16,19,61,151–153,171,178,189,194,207,257,258,283,347 .We have recently observed that the polyclonal anti-a1
antibody, raised by us in a rabbit against the syntheticpeptide corresponding to the amino acids 1–15 of the rata -subunit of GABA receptors, elicits higher percentage1 A
w3 x Žimmunoprecipitation values of the H flunitrazepam 97%. w3 x Ž .vs. 79% and H muscimol 76% vs. 68% binding activ-
ity in the adult rat cerebellum vs. cerebral cortex, whereasw3 xthe immunoprecipitation values for H Ro 15-4513 were
Ž . wcomparable f80% in both the brain regions Mehta andxTicku, unpublished observations . Furthermore, the poly-
clonal anti-b antibody, raised by us in a rabbit against the2w xsynthetic peptide AGLPRHSFGRNA 171 corresponding
to the amino acid residues 382–393 of the rat b -subunit2
( )A.K. Mehta, M.K. TickurBrain Research ReÕiews 29 1999 196–217200
of GABA receptors, elicited higher percentage immuno-Aw3 xprecipitation values of the H flunitrazepam as well as
w3 xH muscimol binding activity in the adult rat cerebellumwvs. cerebral cortex Mehta and Ticku, unpublished observa-
xtions . Aging process is reported to affect the expression ofŽ .GABA receptor subunits mRNA and protein in variousA
w xregions of the rat brain 102,104–106,209,211 as well asradioligand binding to the GABA receptorsAw x75,211,264,265 . However, age-related changes in themRNA expression of a particular subunit does not neces-sarily lead to similar changes in protein or assembly intomature GABA receptors, and it has been proposed thatA
complex regulatory mechanisms of GABA receptor ex-A
pression exist at the transcriptional, translational and post-translational andror assembly levels, which vary with the
w xsubunit and brain area 105 . Recently, insulin has beenreported to increase the number of functional postsynaptic
w xGABA receptor assemblies 340 , whereas chronic GABAA
exposure results in the down-regulation of GABA recep-Aw xtors 108,186,204,263 .
4. Molecular determinants of GABA receptorsA
In an attempt to characterize the molecular determinantsof clinically important drug-targets, several amino acidshave been identified that are important for binding ofligands to the GABA and benzodiazepine sites. It has beenreported that Tyr157, Thr160, Thr 202 and Tyr 205 amino acid
w x 64residues of the b-subunit 3 and Phe of the a -subunit1w x 101284,291 are important for the binding of GABA. HisŽ 102 . w x 200 Ž 201or His in human 70,349 and Gly or Gly in
. w xhuman 248 amino acid residues of the a-subunit, andPhe77, Met130 and Thr142 amino acid residues of the
w xg-subunit 31,33,214,350 are reported to be key determi-nants of the benzodiazepine site of the rat GABA recep-A
tors. Moreover, His102 amino acid residue of the a-subunitinteracts directly with the pendant phenyl group of di-azepam, flunitrazepam, chlordiazepoxide and other 5-
w xphenyl benzodiazepines 193,366 . This His residue isŽ 100.absent in the a -subunit, and it is replaced by Arg Arg4
in the a -subunit of the humanrrat GABA receptors.6 A
These differences in the a - and a -subunits make the4 6
GABA receptor assemblies, derived from the a - orA 4
a -subunit, insensitive to conventional benzodiazepines6wsuch as diazepam, flunitrazepam and clonazepam 152,
x 100153,178,198,258,313 . However, the replacement of Argresidue by Gln100 in the a -subunit of alcohol-sensitive6Ž .alcohol-nontolerant ANT rat cerebellum alters the normaldiazepam-insensitive GABA receptors into diazepam-A
w xsensitive ones 157 . A single amino acid change, from aglutamate to a glycine at position 225 in the a -subunit3
sequence, substantially increases the affinity of the a -3
subunit containing GABA receptors for CL 218872 andAw xzolpidem 248 . Amino acid residues at positions 159
Ž 209. Ž 211.Tyr and 211 Val on the a -subunit of GABA1 A
receptors influence affinities of the ligands for the benzodi-azepine site, thereby suggesting that these amino acids alsoform part of the benzodiazepine binding sitew x 2032,30,32,272,348 . However, mutations of a Gln , the1
amino acid residue located at equal distance to a Gly 2001
and a Thr 206, did not affect the ligand binding, thereby1
indicating that this amino acid does not interact withw xbenzodiazepines and related compounds 272 . It is thus
evident from these studies that three separate domains ofthe a -subunit such as near His101, Tyr159–Thr162 and1
Gly 200–Val211 are involved in the benzodiazepine binding.Similarly, two domains of the g -subunit, Lys41 –Trp82
2
and Arg114–Asp161, have been identified which are re-w xquired for the high affinity benzodiazepine binding 20 .
Although it has been speculated that the co-localization oftwo isoforms of a-subunit and one g-subunit can form twobenzodiazepine sites in the same receptor-assembly pro-vided both the a-subunits are in contact with the g-subunitin the pentamer assembly, this hypothesis is unlikely tohold true due to the fact that the g- and a-subunitscontribute key amino acids to the benzodiazepine site asdescribed above.
Ser 270 as well as Ala291 amino acid residues of thea -subunit, and Ser 265 as well as Met286 of the b -subunit2 1
of GABA receptor are reported to be important aminoA
acids for the action of clinically relevant concentrations ofw x 286the inhalational anesthetic isoflurane 162 . Met amino
acid residue of the b -subunit is important for the potentia-1
tion of the GABA response by the intravenous anestheticagent propofol but this amino acid is not involved in theactivation of the receptor by the high concentrations of
w xpropofol 162 . Furthermore, the extracellular amino-termi-nal half of the a -subunit plays an important role in the1
w xpropofol potentiation of pentobarbital-response 328 . Thew xextracellular N-terminal domain of the a -subunit 86 ,1
273 w x 267His amino acid residue of the a -subunit 86 , His6w x 292residue of the b -subunit 130 , and His amino acid of1
w xthe b -subunit 356 are important molecular determinants3
of the Zn2q binding site on the GABA receptors. Fur-A
thermore, it has been suggested that the Cys–Cys loop ofthe large extracellular NH -terminal domain of the GABA2
r -subunit plays an important role in the receptorrchannel1w xfunctions 175 .
Several amino acids such as b Ser 409, g Ser 327 and1 2S
g Ser 343 have been identified as potential target sites on2L
the GABA receptors for the phosphorylation processAw x164,182,223 in an on-going attempt to investigate therole of the phosphorylation process, mediated throughPKA, PKC and other kinases such as tyrosine kinase, on
wthe modulation of GABA receptor functions 107,116,Ax118,148,164,169,180,182,223,224,296,312,339,344 .
These studies have also revealed that all the major subunitsof GABA receptors contain consensus sites for the phos-A
phorylation process, and this process may be important ina wide range of physiological and pathological processes
in the central nervous system. Recently, it has beenŽ .demonstrated that the b -subunit s of native GABA2r3 A
receptors are phosphorylated in situ, thereby providing adirect evidence that the native GABA receptors are in-A
deed phosphorylated and modulated in situ by endogenousw xkinases 339 . However, the detailed mechanism of phos-
phorylation and its physiological relevance are not pre-cisely known currently.
5. Physiological rrrrr pharmacological role of variousGABA receptor subunits and their receptor assem-A
blies
GABA receptors play a very important role in anxiety.A
Currently, drugs which activate the benzodiazepine site ofGABA receptors are widely used to relieve anxiety.A
However, these classical anxiolytics are full agonists atmost of the GABA receptor subtypes, and have severalA
side effects such as sedation, ataxia, amnesia, tolerence,and physical dependence. During the last decade, attemptshave been made to dissociate their useful therapeutic ef-fects from side effects by developing partial agonists of theGABA receptor subtypes. Unfortunately, the physio-A
logicalrpharmacological role of various native GABAA
receptor assemblies is not yet known. Once this informa-tion becomes available, it will be much easier to synthesizecompounds selective for a particular receptor assembly soas to get a desired therapeutic effect without any majorside effect. Several approaches such as mutation, geneknockout and the inhibition of expression of GABAA
receptor subunits by antisense oligodeoxynucleotides havebeen tried to establish the role of various subunits and theirreceptor assemblies with a limited success so far as de-scribed below and summarized in Table 1.
It has been suggested that motor control is a distinctbehavioral correlate of the a -subunit containing GABA6 A
receptors because the point mutation in the cerebellarŽa -subunit containing GABA receptors replacement of6 A
100 100. ŽArg by Gln results in ethanol intolerance enhanced. Žsensitivity to ethanol in ANT rats alcohol-nontolerant
Table 1Physiologicalrpharmacological role of various subunits of GABA re-A
ceptors
Subunit Possible implications
w xa Behavioral sensitivity to cocaine 2362w x w xa Seizures 44,183,274,322 ; electroconvulsive therapy 43 ;4
w xpre-menstrual syndromes and stress 292,293w xa Motor control 1576
.rats , thereby making the animals highly susceptible to theimpairment of postural reflexes by ethanol and benzodi-
w xazepines 157 . However, there were no differences in thew x w xmetabolism of ethanol 129 , its hypnotic effect 127 ,
w xwithdrawal-hyperexcitability 129 , or reversal of ethanol’sw xeffects by Ro 15-4513 127 in the a -deficient mice vs.6
Žwild-type mice. Furthermore, loss of righting reflex in-.ability to recover normal posture as well as the immobiliz-
ing response to tail-clamp-stimulus in mice anesthetizedwith volatile anesthetics, and sleep-time-response to pento-barbital did not differ between the a -deficient and wild-6
w xtype mice 127 . Interestingly, in the a -deficient cerebel-6
lar granule cells, the d-subunit is selectively degraded asseen by immunoprecipitation, immunocytochemistry andimmunoblot analysis with d-subunit specific antibody,thereby providing an evidence for a specific associationbetween the a and d-subunits in cerebellar granule cells6w x140 . Cerebellar granule cells of the a -subunit deficient6
mutant mice also have severe deficits in the high affinityw3 x w3 xH muscimolr H SR 95531 binding to the GABA sites,
w3 xdiazepam-insensitive H Ro 15-4513 binding to the BZw35 xsites, and furosemide-induced increases in the S t-
Ž .butylbicyclophosphorothionate TBPS binding to the pi-w xcrotoxin-sensitive convulsant sites 185 . DMCM, allo-
pregnanolone and zinc are less efficacious in these mice inw35 xaltering the S TBPS binding in the presence of those
concentrations of GABA that fail to activate the a -sub-1w xunit-containing GABA receptors 185 . Furthermore, theA
inhibition of a -subunit by antisense oligodeoxynucleotide6Ž .ODN in cerebellar granule cells shifts the GABA dose–response curve to the right and significantly increases the
Ž .EC value of GABA a decreased GABA-response ,50
whereas the flunitrazepam-induced potentiation ofw xGABA-activated currents are enhanced 369 . Some of the
above mentioned changes may be adaptive consequencesof altered GABA receptor subunit expression pattern inA
response to the gene knockout. On the other hand, theg -subunit antisense ODN reduces the EC value and2 50
Žshifts the dose–response curve for GABA to the left an.enhanced GABA-response , and decreases the fluni-
trazepam-induced potentiation of GABA-activated currentsw x369 . Furthermore, the bolus infusion of antisense ODN tothe GABA receptor g -subunit is reported to inhibit theA 2
in vivo formation of the benzodiazepine binding sites inw xthe rat cerebral cortex and striatum 146 , and in vivo
formation of the GABA, picrotoxin and benzodiazepinebinding sites are also inhibited in the rat hippocampusw x147 , thereby resulting in an increase in the convulsive
w xthreshold for b-CCM 367 , an inverse agonist for thebenzodiazepine site. Targeted disruption of the g -subunit2
w xgene in mice 101 resulted in the depletion of 94% of thebenzodiazepine sites in the brain of neonatal mice, whilethe number of GABA sites was only slightly reduced.Diazepam was inactive behaviorally in these mice. Fur-thermore, these mice died within a few days after birth,although their birth weight was normal and the major
( )A.K. Mehta, M.K. TickurBrain Research ReÕiews 29 1999 196–217202
peripheral organs, including pituitary, pancreas and adrenalgland which are known to express GABA receptors, didA
not reveal any pathological changes. The surviving mu-tants exhibited excessive hyperactivity, impaired grasping,impaired righting reflexes and abnormal gait. None ofthese mutant mice survived beyond P18, although feeding
w xwas apparently not impaired 101 . Recently, mice devoidw xof the g -subunit have been produced 128 . These mice2L
are viable, healthy, and overtly indistinguishable from thewild-type. However, there was up-regulation of the g -2S
subunit as a result of compensatory mechanism in thesew xmutant mice devoid of the g -subunit 128 . Potentiation2L
of the GABA currents by ethanol as well as severalbehavioral effects of ethanol such as sleep time, anxiolysis,acute function tolerance and chronic withdrawal hyperex-citability were not altered in these mutant mice, therebysuggesting that the g -subunit is not required for the2L
modulatory effects of ethanol at the GABA receptorsAw x128 . These results contradict the previous view that theg -subunit of GABA receptor is a critical molecular site2L A
w xof action for ethanol 335 but support the reports indicat-ing that the presence of g -subunit is not an absolute2L
requirement for the potentiation of GABA-induced re-w xsponses by ethanol 190,285 . Mice deficient in the b -sub-3
w xunit of GABA receptor usually die as neonates 126 . TheA
mice that survive are runted until weaning, and have areduced life span. These mutant mice are fertile but moth-
w xers fail to nurture offspring 126 . Brain morphology isgrossly normal but these mice are hyperactive, hyperre-sponsive to human contact and often run continuously in
w xtight circles 126 . When held by tail, these mutant micehold all the paws in like a ball. They also exhibit tremor,
w xjerky gait, cleft palate 53,54,126 and Angelman syn-w xdrome 179,225,262,267 , an inherited neurobehavioral
disorder that is characterized in humans by seizures, ataxia,stiff jerky movements, absence of speech and severe men-tal retardation. Angelman syndrome in the b -subunit-defi-3
w xcient mice 179,225,262,267 is consistent with the reportthat the gene encoding the GABA receptor b -subunitA 3
maps to the Angelman syndrome region on chromosomeŽ .15, and deletion of this gene GABRB3 is found in the
w xAngelman syndrome patients 337 . Furthermore, based ongene knockout study, it has been reported that the b -sub-3
unit is important in the mediation of the immobilizingresponse due to the volatile anesthetic agents such as
w xenflurane and halothane 255 , whereas the loss of rightingreflex due to the volatile anesthetic agents and pento-
w xbarbital is not affected by the absence of b -subunit 255 .3
However, the loss of righting reflex due to midazolam andetomidate is attenuated by the absence of the b -subunit3w x255 . Administration of the antisense oligodeoxynu-cleotide to the GABA receptor a - or b -subunit intoA 2 3
striatum results in the increased behavioral sensitivity tococaine in rats, thereby suggesting the role of these sub-
w xunits in determining cocaine-sensitivity 236 . Although itwas thought that the deletion of a - and g -subunits may5 3
w xresult in neurological phenotype 225 , this possibility wasruled out based on the observation that mutation of thesesubunits does not result in any overt neurological pheno-type in mice and thus does not provide useful animal
w xmodels for the Angelman syndrome in humans 54,225 .The above-mentioned studies suggest that the antisenseoligodeoxynucleotide treatment and gene-knockout tech-niques are useful tools for studying the regulation ofreceptor structurerfunction and revealing the physiologicaland pharmacological significance of various GABA re-A
ceptor subunits and their receptor assemblies.In an attempt to investigate the role of a -subunit in4
seizures, it has been shown that a -subunit mRNA is4
increased in the dentate gyrus at 4 h after the fifth amyg-w xdaloid kindled seizure 44 . However, another group of
investigators did not find any change in the a -subunit4
mRNA levels at 24 h after the sixth fully kindled seizurew xelicited by Schaffer collateral stimulation 144 . Role of
the GABA receptor a -subunit in kainic acid-inducedA 4
seizures in rats has also been investigated by severalresearchers because these seizures in rats represent ananimal model for human temporal lobe epilepsy. Theneuropathological events uderlying these seizures include
Ž .acute limbic seizures status epilepticus followed by neu-rodegeneration in the hippocampus, thereby ultimatelymanifesting the spontaneous recurrent seizures after about3 weeks. Interestingly, recurrent spontaneous seizures fol-lowing the administration of kainic acid result in the
w xup-regulation of a -subunit mRNA 322 . Furthermore,4
the a -subunit immunoreactivity is also reported to be4
increased in the rat hippocampus at 30 days after thew xadministration of kainic acid 274 . Electroconvulsive shock
Ž .ECS treatment has been shown recently to increase thew3 xa -subunit mRNA and the diazepam-insensitive H Ro4
15-4513 binding in the dentate gyrus, thereby suggestingthat the a -subunit of GABA receptor could be impli-4 A
cated in the clinical effects of electroconvulsive therapyw x43 . Furthermore, the inhibition of a -subunit by the4
antisense oligodeoxynucleotide is reported to prevent theincreased seizure susceptibility observed following proges-
w xterone withdrawal 292 . Therefore, it has been speculatedthat the a -subunit of GABA receptor plays an important4 A
role during periods of fluctuations in the levels of endoge-nous neuroactive steroids associated with the menstrualand pregnancy cycles in females and during stress in males
w xas well as females 292,293 . A long-lasting kindling phe-Ž .nomenon associated with chronic intermittent ethanol CIE
treatment is also reported to result in an increase in thea -subunit mRNA levels in the thalamus, dentate gyrus,4
CA1 and CA3 regions of hippocampus, and layers II andIII of the cortex, thereby further supporting the role of
w xa -subunit of GABA receptors in seizures 183 . Al-4 A
though the genetic studies have revealed that the differ-ences between the highly prone DBAr2J mice to juvenileaudiogenic seizures and the seizure-resistant C57BLr6J
w xmice are due to several genetic factors 226 yet these two
w xstrains of mice do not differ in the a - 342 , b -1 – 3 1 – 3w x w x w x w x143 , d- 341 , g - 343 and g -subunits 156 . However,1 2
the comparison of a -subunit of GABA receptors has4 A
not been investigated in these mice.
6. Interaction of drugs with GABA receptorsA
Several drugs such as benzodiazepines, barbiturates,neurosteroids, ethanol, some of the anticonvulsants, andgeneral anesthetics interact with GABA receptors so as toA
elicit their pharmacological effects. The role of GABAA
receptors in the action of these categories of drugs hasbeen reviewed from time to time by us and other re-
w xsearchers 282,308–310,313–316,347 . In this section, wewould summarize the recent developments in this area.
6.1. Anxiolytics
In an attempt to dissociate the desired therapeutic effectof anxiolytics acting at the benzodiazepine site of GABAA
receptors from their side effects, several new compoundsw x wsuch as imidazenil 7,60,98,100,277,306 , abecarnil 155,
x w196,198,233,234,276,278,297,325 , bretazenil 27,84,x w111,155,191,198,219,245,249 , and divaplon 82,93,137,
x155 have been synthesized and evaluated for their phar-macological effects. Some of these compounds are partialagonists at some GABA receptor subtypes and full ago-A
nists at others as is the case with abecarnil which is apartial agonist at the a b g GABA receptor assembly5 2 2 A
and a full agonist at a b g GABA receptor assembly3 2 3 Aw x155 . On the other hand, divaplon is a partial agonist atboth a b g and a b g GABA receptor assemblies3 2 3 5 2 2 Aw x155 , and imidazenil has low efficacy at a broad spectrum
w xof GABA receptor subtypes 100 . As a result of theseA
features, these compounds are devoid of many, if not all,side effects associated with conventional anxiolytic benzo-diazepines because the lower intrinsic efficacy of theGABA receptor partial agonists is probably sufficient toA
maintain the low efficacy responses such as anxiolytic andanticonvulsant responses, but insufficient to induce seda-tion, myorelaxation, dependence and ethanol-potentiation,which require high fractional receptor occupancyw x78,94,138,238 . Accordingly, abecarnil is a potent anxi-olytic and elicits no or only weak effects in tests of motorincoordination and muscle relaxation, and has a relativelyweak ability to potentiate the effects of ethanol and barbi-
w xturates in contrast to diazepam 297 . The therapeuticsafety, defined as the ratio between the anticonvulsantefficacy and the muscle relaxant action, is more favorablefor abecarnil as compared to diazepam in rodents and
Ž . w xbaboon Papio papio 325 . Abecarnil also elicits potentanticonflict and taming effects with little sedative and
w xataxic effects in primates 233 . Chronic administration ofabecarnil was found to elicit persistent anxiolytic and
w xanticonvulsant effects without any amnesia in rats 234 ,
and it did not induce tolerance or withdrawal syndromes inw xmice 278 . Interestingly, imidazenil, a potent anxiolytic, is
devoid of sedation, ataxia and ethanol-potentiation, andblocks the sedative and ataxic effects of diazepam in ratsw x100 . It also completely attenuates the benzodiazepine-in-
w xduced cognition deficit in monkeys 306 . Furthermore, itcauses only low tolerance and dependence liabilities in ratsw x7 . Chronic treatment with a pharmacologically effectivedose of imidazenil failed to induce tolerance to the effectsof this drug on GABA receptor function in mouse brainAw x98 . Bretazenil elicits potent anticonflict and anticonvul-sant activity but it results in a lower degree of sedation,
w x w xtolerance 111 , dependence liability 27,219 , and etha-w xnol-potentiation 191 as compared to conventional benzo-
diazepines. Likewise, divaplon elicits potent anticonflictw xactivity, and it results in lower sedation 93 and tolerance
w x82,137 as compared to diazepam. According to a recentreport, chronic exposure of primary cultured cerebellargranule cells to the agonists of the benzodiazepine siteresults in a reduction of the a -subunit protein expression,1
and the magnitude of response depends upon the efficacyw xof these agents 139 . Therefore, it has been suggested that
this model may serve as an indicator of benzodiazepineagonist efficacy with the ability to differentiate between
w xpartial agonists of the benzodiazepine site 139 . Chronictreatment with conventional benzodiazepines, having fullagonistic effect, decreases the mRNA levels of the a - and1
w xg -subunits 123,145 , and increases the levels of the a -2 3w xand a -subunit mRNA in the rat brain 229 . Furthermore,6
chronic treatment with the conventional benzodiazepinesdecreases the efficacy of barbiturates as well as
w xagonistsrinverse agonists of the benzodiazepine site 133 ,uncouples the GABA and barbiturate sites from the benzo-
w xdiazepine site 132 , and decreases coupling between thebenzodiazepine site and GABA receptor-gated chlorideA
w xchannels 132 . These changes may be responsible fortolerance and dependence following chronic treatment withconventional benzodiazepines.
6.2. AnticonÕulsants
Loreclezole, an anticonvulsant, is reported to interactw xwith GABA receptors 334 . It does not require theA
presence of either an a- or a g-subunit of GABA recep-A
tor, but is highly selective for GABA receptor assembliesAw xcontaining the b -subunit versus b -subunit 334 . It has2r3 1
a negligible affinity for the benzodiazepine site, and theanticonvulsant effect could be reversed by some inverseagonists of the benzodiazepine site but not by Ro 15-1788w x59 . The interaction of loreclezole with GABA receptorsA
has also been demonstrated in recombinant receptor sys-w xtem 85,351 . Tiagabine, another anticonvulsant agent
w x240,300 , elicits its effects as a result of inhibition of thew xGABA uptake 25 , and it has a potential utility in the
treatment of chronic seizure disorders such as generalizedclonic–tonic epilepsy, photomyoclonic seizures, myoclonic
( )A.K. Mehta, M.K. TickurBrain Research ReÕiews 29 1999 196–217204
w xpetit mal epilepsy and complex partial epilepsy 300 .Ž .Vigabatrin, i.e., g-vinyl GABA GVG , is an inhibitor of
GABA aminotransferase, and it crosses the blood–brainŽ .barrier unlike GABA , thereby resulting in a dose-depen-
dent long-lasting enhancement of the GABA concentrationw xin the brain 114,174 . It is an effective treatment for
partial complex seizures in pediatric and adult patientswith pharmacoresistance to the conventional antiepileptic
w xdrugs 38,273 . Gabapentin, a structural analogue ofGABA, was synthesized with the idea that it would elicitthe physiological actions of GABA in the brain. Althoughgabapentin, unlike GABA, crosses the blood–brain barrier
w xand elicits anticonvulsant effects 11,62 , it does not inter-act with GABA or GABA receptors. Moreover, it is notA B
converted metabolically into GABA or agonist of GABA-receptor and it is not an inhibitor of GABA uptake or of
w xGABA degradation 333 . However, it has been reported toincrease the GABA turnover by activation of glutamate
w xdecarboxylase 304 . It has also been reported recently thatgabapentin increases the levels of GABA in the brains of
w x wepileptic patients 239 and relieves partial epilepsy 38,x w x w x273 . Gabapentin 188 and vigabatrin 66 have been
reported recently to attenuate the cocaine withdrawal andcraving by potentiating the GABAergic transmission. Po-tentiation of the GABAergic system is also thought to beinvolved in the anticonvulsant effects of valproate, barbitu-rates, benzodiazepines and felbamate in addition to theability of these drugs to block the voltage-dependent Naq
w xchannels andror glutamatergic mechanisms 174,199,310 .Ž .GABA receptor agonists such as THIP gaboxadolA
w x w x92,201 and progabide 203,241 are reported to elicitanticonvulsant effect in experimental animals. Topiramate,an anticonvulsant drug effective in treating simple orcomplex partial seizures with or without secondarily gener-
w xalization 261,266,279,303 , is reported to potentiate theeffect of GABA on chloride-flux mediated through GABAA
w xreceptors 28 , besides its interaction with excitatory aminow xacid receptors 49 and voltage-gated sodium channels
w x48,370 . However, the exact molecular mechanism of itsanticonvulsant effect is not yet established.
6.3. General anesthetics
Several general anesthetics of diverse structure such asisoflurane, enflurane, halothane, barbiturates, etomidate,steroid anesthetics and propofol have been reported tointeract with GABA receptors at clinically relevant con-A
w xcentrations 9,46,112,115,141,172,237,327 . Their exactbinding site on GABA receptor and subunit requirementA
are not yet fully established. Mutation of the residuesŽ . Ž .within either a S270 or A291 or b S265 or M2862 1
subunits of GABA receptor is reported to cause insensi-A
tivity to clinically relevant concentrations of the inhala-w xtional anesthetic isoflurane 162 . The b-subunit of GABAA
receptor appears to contribute to the direct actions ofŽ .propofol 2,6-diisopropylphenol , an intravenous general
w xanesthetic which is used clinically these days 58,268 . AŽ .point mutation in the b -subunit M286W abolished1
potentiation of the GABA response by propofol but it didnot alter direct activation of the receptor by high concen-
w xtrations of propofol 162 . Furthermore, the a- and g -2L
subunits influence both the direct and modulatory actionsw xof propofol on GABA receptor function 167 . PropofolA
is also reported to potentiate the pentobarbital-inducedcurrent, and the a -subunit of GABA receptor is neces-1 A
w xsary for this modulatory action 328 . It seems that theextracellular amino-terminal half of the a -subunit is suffi-1
cient to support the propofol potentiation of pentobarbital-w xresponse 328 . However, the direct activation of GABAA
receptor by propofol has been suggested to be a distinctw xprocess mediated through a distinct site 328 . Interest-
ingly, propofol, unlike conventional benzodiazepines, po-tentiated the a b g GABA receptor-mediated responses4 1 2 A
comparable to those of the a b g GABA receptor1 1 2 Aw xassembly 336 . However, it failed to elicit a direct activa-
w xtion of the a b g GABA receptors 336 .4 1 2 A
6.4. Barbiturates
Barbiturates have been known to elicit their pharmaco-logical effects via the GABA receptors. These drugsA
potentiate the effect of GABA at lower concentrations andactivate the receptor directly at higher concentrations
w xthrough two distinct sites 3 . The a-subunit is reported toŽ .influence the degree of efficacy but not affinity of pento-
w xbarbital-induced potentiation of the GABA responses 307 .However, the b-subunit does not have any marked effecton the affinity or efficacy of the potentiating responsew x110 . Recently, pentobarbital has been shown to activatethe homomeric GABA receptors containing b -subunit inA 3
w xXenopus oocytes 328 . The a-subunit is reported to influ-ence the affinity as well as efficacy of direct activation ofGABA receptors by pentobarbital at higher concentra-A
w xtions 307 . Interestingly, pentobarbital, unlike conven-tional benzodiazepines, potentiates the GABA responsesmediated through a b g as well as a b g GABA4 1 2 6 1 2 A
w xreceptors 336 . Moreover, pentobarbital was able to acti-vate the a b g GABA receptors directly, but it failed to6 1 2 A
elicit a similar effect at the a b g GABA receptors4 1 2 Aw x336 . Chronic pentobarbital administration leads to toler-ance and physical dependence. The d-subunit mRNA inthe cerebellum is upregulated in the pentobarbital-tolerantmice, and is down-regulated in the pentobarbital-withdrawn
w xmice 173 . These observations implicate the role of thed-subunit of GABA receptor in the action of pento-A
barbital. Furthermore, the a -subunit mRNA in the rat6
cerebellum is upregulated in the pentobarbital-tolerant ani-mals, thereby resulting in an enhancement of the di-
w3 x w xazepam-insensitive H Ro 15-4513 binding 136 . Thepentobarbital-dependent rats are also reported to expressthe increased levels of the a - and g -subunit mRNA of1 2
GABA receptor, whereas the decreased levels of mRNAA
of these subunits are observed in the pentobarbital-tolerantw xrats 320,321 . The expression of b mRNA is increased in1
the hippocampal pyramidal cells of CA1 and CA2, but notin hippocampal CA3–4 or cerebral cortex, following
w xchronic treatment of rats with pentobarbital 358 . Chronictreatment of primary cultured cerebellar granule cells withpentobarbital decreased the GABA receptor subunitA
g rg mRNA ratio but this effect is not mediated via2L 2Sw xdirect activation of the receptor itself 326 . Neither the
mechanism nor the impact of decreasing the g rg2L 2S
mRNA ratio by pentobarbital is known, but changes in theratio could produce a decreased phosphorylation of theGABA receptor with subsequent alterations to GABAA A
w xreceptor function 326 .
6.5. Ethanol
Ethanol shares several pharmacological actions with thebenzodiazepines and barbiturates, and several of its effects
ware mediated through GABA receptors 1,166,200,Ax205,301,302,311,313 . On the other hand, GABA recep-B
tors do not seem to be involved in the pharmacologicalw xeffects of ethanol 203 . It has been suggested that ethanol
sensitivity is correlated with the presence of BZ I bindingŽw3 x . w xsites H zolpidem binding sites 26,51,52 as well as
with the a -, b - and g -subunits of GABA receptors1 2 2 Aw x51,71 . Over the last decade, effects of chronic ethanoladministration and its abrupt withdrawal on the mRNAand polypeptide levels of GABA receptor subunits haveA
been investigated in several brain regions in an attempt toexplain the mechanisms underlying its dependence andtolerance. Chronic ethanol administration is reported toreduce the levels of a -, a -, a -subunit mRNA and1 2 5
wpolypeptide in cerebellum and cerebral cortex 29,41,x63,210,212,217,220 . However, chronic administration of
ethanol or its abrupt withdrawal following chronic admin-istration did not result in the down-regulation of theGABA receptor assemblies containing a -subunit in bothA 1
w3 xthe brain regions as determined by the H muscimol,w3 x w3 x w3 xH flunitrazepam, H Ro 15-4513 and H zolpidembinding to the immunoprecipitated GABA receptors us-A
wing a -subunit-specific polyclonal antibody Mehta and1xTicku, unpublished observations . In contrast to the de-
crease in the mRNA and polypeptide levels of the a -sub-1
unit by chronic ethanol administration, the a -subunit5w xmRNA levels are increased in the rat hippocampus 41 .
w x w xSimilarly, the a -, g -, g - 65 , a - 212 and b -sub-4 1 2S 6 2r3w xunits 213 mRNA levels increase in the ethanol-dependent
animals. Furthermore, the polypeptide levels of the a -,4
b -, and g -subunits of GABA receptors are also in-2r3 1 A
creased in the cerebral cortex of ethanol-dependent andw xethanol-withdrawn rats 63 . However, chronic administra-
tion of ethanol did not alter the mRNA levels of g -, g -2L 3w xand d-subunits 65 as well as the polypeptide levels of
w xg -subunit 63 in cerebral cortex. During the behavioral2
peak of ethanol withdrawal, mRNA levels of the a -, a -,1 4
and g -subunits revert nearly to control levels, whereas2S
significant increase in the b -, b -, and g -subunit mRNA2 3 1w xlevels are observed in the rat cerebral cortex 64 . Chronic
Ž .intermittent ethanol CIE treatment in rats leads to anincrease in the a -subunit mRNA levels in hippocampus,4
w xcerebral cortex and thalamus 183 . A new in vitro modelof CIE using cultured cortical neurons has been developedfor studying the biochemical and molecular mechanismsunderlying the CIE-induced kindling-like phenomenon ob-
w xserved in humans 134 . In the postmortem frontal cortexof human alcoholics, the GABA receptor a -, a -, andA 1 4
b -subunit peptide expression did not differ as compared2r3w xto control group 215 . Furthermore, no differences in
GABA receptor a - or a -subunit mRNA were found,A 1 4
while higher levels of b -subunit mRNA were found in3w xhuman alcoholics 215 . These results suggest that the
effects of chronic ethanol exposure in human alcoholicsdiffer from the rat model of ethanol-dependence.
w3 xChronic ethanol treatment increases the H Ro 15-4513w x w xbinding in the rat brain 208 and mouse cerebellum 14 ,
Ž .and alters the behavioral effects of Ro 15-4513 sarmazenilw x13,202 , a partial inverse agonist of the benzodiazepinesite which is reported to antagonize the pharmacologicaleffects of low doses of ethanol mediated through GABAA
w x w3 xreceptors 200,309,313 . The H Muscimol binding sitesw xare increased in the alcoholic human cerebral cortex 318w xand superior frontal gyrus of noncirrhotic alcoholics 67 .
There is a possibility that phosphorylation plays a role inthe biochemical events following chronic administration of
w xethanol and its abrupt withdrawal 344 . Selectively bredŽ .mouse lines SS: short sleep; LS: long sleep and rat lines
wAT: alcohol-tolerant or alcohol-insensitive; ANT:alcohol-nontolerant or alcohol-sensitive; alcohol-preferring
xrats and alcohol-nonpreferring rats have also implicatedwthe GABA receptors in the action of ethanol 1,77,A
x299,329 . Ethanol potentiates the GABA responses of cer-tain brain regions in LS mice but not in SS mice, which isconsistent with the much longer ethanol-induced sleep
w xtime in the LS mice 1 . On the other hand, the alcohol-sensitive ANT rats differ from the alcohol-insensitive ATrat lines by a critical point mutation of the a -subunit of6
Ž 100 100GABA receptors Arg residue is replaced by Gln inA.the ANT rat line , thereby altering the normal diazepam-
w xinsensitive receptors into diazepam-sensitive ones 157 .Although the ANT rats are more sensitive to the motor
w ximpairment induced by ethanol 77 or benzodiazepinesw x122 as compared to the AT rats, there is no substantialdifference in the sensitivity to the hypnotic doses of ethanolw x76,161 . The ANT rats exhibit a diminished stress-re-sponses to a variety of stressors, even in the non-intoxi-
w xcated state 323 . Furthermore, Ro 15-4513 has almost twotimes more affinity for the ANT rat cerebellum versus the
w xAT rat cerebellum 329 . Alcohol-preferring rats are re-w xported to be more active in the open field 142,338 , and
are innately anxious as assessed by the less time spent onthe open arms as compared to alcohol-nonpreferring rats
( )A.K. Mehta, M.K. TickurBrain Research ReÕiews 29 1999 196–217206
w x299 . Benzodiazepine receptor antagonists are reported tomodulate the behavioral actions of ethanol in the alcohol-
w xpreferring and -nonpreferring rats differentially 142 . Al-Ž .cohol-preferring Fawn-Hooded FH rats are reported to
w3 xpossess a significantly higher density of H zolpidembinding sites as compared to alcohol-nonpreferring
Ž .Wistar-Kyoto WKY rats in brain regions involved inreward processes such as cortical regions, substantia nigra
w xpars reticulata and the ventral pallidum 42 . Selectivelybred rodent lines thus offer a very useful tool to investigatethe pharmacology of ethanol.
6.6. Neurosteroids
Several neurosteroids have been shown to interact withw xthe GABA receptors 184,206,251 , as was first demon-A
strated for a synthetic neurosteroid anesthetic alphaxalonew x117 . These drugs interact with a distinct neurosteroid-bi-nding site, different from the barbiturate-site, on GABAA
w xreceptors 97,324 . Furthermore, the presence of multipledistinct steroid recognition sites or conformational states
w xon GABA receptors has also been suggested 120,221 .A
However, it has been suggested recently that dehy-Ž .droepiandrosterone sulfate DHEAS , a neurosteroid, binds
to the picrotoxin site of GABA receptors, whereas dehy-AŽ .droepiandrosterone DHEA does not interact with GABAA
w xreceptor complex 294 . Neurosteroids, in general, interactwith the GABA receptor assemblies composed of a -,A 1
w xb -, and g -subunits or b -subunits alone 251 . Modula-1 2 2
tion of the GABA response by steroids requires the pres-ence of b-subunit, and does not depend on the presence of
w xa g-subunit unlike the benzodiazepine site 251 . However,the substitution of a g -subunit for a g -subunit greatly1 2
w xenhances the sensitivity to neuroactive steroids 250,252 .On the other hand, d- and ´-subunits inhibit the neuros-
w xteroid modulation of GABA receptors 57,368 , whereasA
the type of a-subunit isoform does not seem to play amajor role in determining the neurosteroid efficacy or
w xpotency as a positive modulator 250 . The a -subunit4
suppression prevents withdrawal effects of progesteroneŽ .which include symptoms of premenstrual syndrome PMS
w xsuch as anxiety and seizure susceptibility 292 . The pseu-dopregnancy paradigm has been suggested as a usefulmodel for periods of endogenous neurosteroid withdrawalsuch as PMS and postpartum or postmenopausal dysphoriaw x293 which are associated with increased emotional liabil-
w xity and benzodiazepine-insensitivity 47,292,293 . Chronicneurosteroid treatment decreases the mRNA levels of a -,2
w xa -, b - and b -subunits 360 , and results in a heterolo-3 2 3
gous uncoupling between the GABA, barbiturate, neuros-w xteroid and benzodiazepine site 87,362 , and a reduced
efficacy of GABA, the benzodiazepine site ligands andneurosteroids at the GABA receptors in a heterologousA
w xmanner 361 . Recently, neurosteroids are reported to bemore effective in inhibiting bicuculline-induced seizures in
the ethanol-withdrawn rats and in potentiating the GABAA
receptor-mediated chloride uptake in synaptosomes pre-pared from the cerebral cortex of ethanol-withdrawn rats
w xas compared to control group 64 . Interestingly, the a -6
containing recombinant receptors are more sensitive to thew35 xinhibition of S TBPS binding by 5a-pregnan-3a-ol-20-
w xone than those containing the a -subunits 160 . 5a-Preg-1
nan-3a-ol-20-one is a neurosteroid and a natural activemetabolite of progesterone which is also known as allo-pregnanolone or allopregnan-3a-ol-20-one or 3a-hydroxy-5a-pregnan-20-one, and is almost equi-potent to benzodi-azepines in potentiating the GABA receptor-mediatedA
w xchloride ion flux 222 . It has hypnotic and anxiolyticw xeffects 50,125 which correlate with its efficacy on
GABAergic transmission in the CNS, and potentiates thew3 x Žbinding of H flunitrazepam a ligand for the benzodi-. w3 x Žazepine-site , and H muscimol a ligand for the GABA-.binding site in the rat cerebral cortex, cerebellum and
w xhippocampus 206 . 5a-Pregnan-3a-ol-20-one also eliciteda biphasic response, i.e., it potentiated the binding ofw35 x Ž .S TBPS a ligand for the picrotoxin-site at lower con-centrations and inhibited the binding at higher concentra-tions in the rat cerebral cortex, cerebellum and hippocam-
w xpus 206 . Furthermore, it is more efficacious in enhancingw3 x w3 xthe binding of H flunitrazepam and H muscimol in the
cerebellum of ethanol-dependent rats as compared to con-w xtrol group 206 , and is more efficacious in inhibiting thew35 xbinding of S TBPS in the hippocampus of ethanol-de-
w xpendent and ethanol-withdrawn rats 206 , thereby suggest-ing that the neurosteroid binding site associated withGABA receptors plays an important role during ethanol-A
w xdependence and ethanol-withdrawal 206 . However, 5a-pregnan-3a-ol-20-one has poor oral availability. To im-prove bioavailability, a new synthetic neuroactive steroid
Ž .ganaxolone 3a-hydroxy-3b-methyl-5a-pregnan-20-onehas been synthesized and is currently under clinical trials.It is a potent and efficacious anticonvulsant agent for themanagement of generalized absence and partial seizuresw x35 as well as for convulsions due to cocaine poisoningw x96 . It is more potent and efficacious as compared tovalproate and diazepam in blocking the development of
Ž . w xpentylenetetrazole PTZ -kindled seizures 95 . Recently, itwas reported that ganaxolone is superior to valproate,ethosuximide, clonazepam, diazepam, and phenobarbital inpreventing the PTZ-induced convulsions and the behav-ioral effects of PTZ including its depressant effects onlocomotor activity and rearing in mice, thereby suggestingthat ganaxolone may provide additional benefits in thetreatment of epilepsy by controlling anxiety, mood changesand other behavioral alterations associated with pre-seizure
w xactivity 15 .
6.7. Miscellaneous
The divalent cation zinc is reported to block the GABAA
receptor-mediated responses in a non-competitive and volt-
w xage-dependent manner 68,86,130,163,168,289,290,356 .Receptor assemblies derived from a combination of a- andb-subunits are highly sensitive to zinc-blockade but thosecontaining a g-subunit are almost resistant to blockade by
w xzinc 68,290 . In other words, GABA receptors with aA
benzodiazepine-site are resistant to blockade by zinc whilethose devoid of the benzodiazepine-site are sensitive toblockade by zinc in most instances. The extracellular
w x 273N-terminal domain of the a -subunit 86 , His amino1w x 267acid residue of the a -subunit 86 , His residue of the6
w x 292b -subunit 130 , and His aminoacid of the b -subunit1 3w x356 are reported to be important determinants of theZn2q binding site on the GABA receptors. ThyroidA
Ž .hormones such as L-triiodothyronine T3 and L-thyroxineare also reported to interact with the GABA receptorsAw x40,192 , and it has been suggested that the a -subunit1
w ximparts T3 sensitivity to the GABA receptors 40 . Al-A
though somatostatin-14, a biologically active tetradecapep-tide, is known to mediate its biological actions through the
w xG protein-coupled membrane receptors 332 , its interac-tion with GABA receptor complex has also been reportedAw x74,91,331 . Chronic administration of antipsychotic drugssuch as clozapine and olanzapine is reported to decreasethe rat cortical and limbic GABA receptors probablyA
in response to the increased GABA release followingthe blockade of multiple neurotransmitter receptors onGABAergic interneurons, and it has been suggested thatthese antipsychotic drugs would assist in substantiallyreversing an under-active GABAergic system in schizo-
w xphrenia 79 . The pineal gland hormone melatoninw x w x45,170,187,227 , lanthanum 135,271 , g-butyrolactones
w x w xas well as g-thiobutyrolactones 124 , penicillin 131 ,w xantihelminthic compound ivermectin 165,244 , pesticide
w xcompounds lindane, endrin and dieldrin 317 , anxiolyticranticonvulsant compounds chlormethiazole and
w xtrichloroethanol 113,218,235 , polyamines such as sper-w xmine and spermidine 99 , and antidepressants such as
w xamoxapine and mianserin 295 have been reported tointeract with GABA receptors but the exact site of actionA
of these drugs on the GABA receptors and their subunitsA
requirement are not known currently.
7. Concluding remarks
The heterogeneity of GABA receptors in the brain isA
much larger than originally thought. There is a largenumber of different GABA receptor subunits with dis-A
tinct regional distribution. The pentameric GABA recep-A
tor assembly can be derived from a permutation andcombination of two, three, four, or even five differentsubunits. Composition of various GABA receptor assem-A
blies can differ not only in different parts of the brain or indifferent cells but also in the same cell. Although the a -,1
b -, and g -subunits co-exist in many native GABA2r3 2 A
receptors, the composition of major receptor assemblies invarious regions of brain has not been characterized fully.Radioligand binding to the immunoprecipitated receptorsusing subunit-specific antibodies, co-localization studiesusing immunoaffinity chromatography, immunocytochem-istry, and the recombinant receptors system are useful toolsto provide this information in future. Furthermore, charac-terization of molecular determinants of clinically importantdrug-targets is under way. Based on new informationavailable so far with these techniques, better anxiolyticsare being developed. These drugs are partial agonists forGABA receptor subtypes, and have fewer and less severeA
side effects as compared to conventional benzodiazepineanxiolytics. However, the progress in this area is hinderedby the fact that we still do not know the composition andphysiologicalrpharmacological significance of various na-tive GABA receptor assemblies. Various experimentalA
approaches such as mutation, gene knockout and inhibitionof expression of GABA receptor subunits by antisenseA
oligodeoxynucleotides are likely to provide answers tosome of these questions, and help us in developing newerdrugs devoid of major side effects associated with cur-rently available anxiolytics as a result of their betterselectivity for various GABA receptor assemblies.A
Acknowledgements
We thank Ms. Elena Wright for her valuable help inpreparing this review article, and Ms. Sadie Phillips forexcellent secretarial help. Support for research work fromauthors’ laboratory and for preparing this review articlewas provided by the NIH-NINCDS grants NS15339 andNS24339, and NIH-NIAAA grants AA04090 andAA10552.
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