gamma2L Subunit Research Articles

Identification of a domain in the δ subunit (S238‐V264) of the α4β3δ GABAA receptor that confers high agonist sensitivity

We have expressed the alpha4beta3delta and alpha4beta3gamma2L subtypes of the rat GABAA receptor in Xenopus oocytes and have investigated their agonist activation properties. GABA was a more potent agonist of the alpha4beta3delta receptor (EC50 approximately 1.4 micromol/L) than of the alpha4beta3gamma2L subtype (EC50 approximately 27.6 micromol/L). Other GABAA receptor agonists (muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, imidazole-4-amino acid) displayed similar subtype selectivity. The structural determinants underlying these differences have been investigated by co-expressing chimeric delta/gamma2L subunits with alpha4 and beta3 subunits. A stretch of amino acids in the delta subunit, S238-V264, is shown to play an important role in determining both agonist potency and the efficacies of full or partial agonists. This segment includes transmembrane domain 1 and the short intracellular loop that leads to the second transmembrane domain. The effects of the competitive antagonists, bicuculline and SR95531, and the channel blocker, picrotoxin, were not significantly affected by the incorporation of chimeric subunits. As the delta and gamma2L subunits have not been previously implicated directly in agonist binding, we suggest that the effects are likely to arise from changes in the transduction mechanisms that link agonist binding to channel activation.

Dual Potentiating and Inhibitory Actions of a Benz[e]indene Neurosteroid Analog on Recombinant α1β2γ2 GABAA Receptors

Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.

Multiple actions of propofol on alphabetagamma and alphabetadelta GABAA receptors.

GABAA receptors are predominantly composed of alphabetagamma and alphabetadelta isoforms in the brain. It has been proposed that alphabetagamma receptors mediate phasic inhibition, whereas alphabetadelta receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABAA receptors; however, the effects of propofol on the kinetic properties of alphabetagamma and alphabetadelta receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat alpha1, alpha6, beta3, gamma2L, or delta subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 microM) increased GABA concentration-response curve maximal currents similarly for both alpha1beta3gamma2L and alpha6beta3gamma2L receptors, but propofol increased those for alpha1beta3delta and alpha6beta3delta receptors differently, the increase being greater for alpha1beta3delta than for alpha6beta3delta receptors. Propofol (10 microM) produced similar alterations in alpha1beta3gamma2L and alpha6beta3gamma2L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both alpha1beta3delta and alpha6beta3delta receptors, but the enhancement was greater for alpha1beta3delta receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of alpha1beta3delta receptor currents but did slow deactivation of alpha6beta3delta receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of gamma2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of delta subunit-containing receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain.

Neuroactive steroids have multiple actions to potentiate GABAA receptors.

The effects of neuroactive steroids on the function of GABAA receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with alpha1 beta2 gamma2L subunits. Activity was elicited with a half-maximal (50 microM) concentration of GABA. Two steroids were studied in detail: ACN ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times. When clusters of openings were elicited in the absence of steroid, the open time distribution contained three components. ACN produced concentration-dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nM ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nM ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nM B285) but increased the duration only at concentrations close to 10 microM. The differences in the actions of these two steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nM ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC50 concentrations (about 40 nM ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 microM ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA. Mutation of the carboxy terminus of the gamma2 subunit, but not the alpha1 or beta2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there is more than one distinguishable steroid recognition site on the GABAA receptor.

On the mechanism of a mutated and abnormally functioning gamma-aminobutyric acid (A) receptor linked to epilepsy.

A recent report indicates that a lysine-to-methionine mutation (K289M) in the gamma2 subunit of a human gamma-aminobutyric acid neurotransmitter receptor, the GABA(A) receptor, is linked to generalized epilepsy with febrile seizures [Baulac et al. (2001) Nat. Genet. 28, 46-48]. This mutation caused a decreased current response to GABA [Baulac et al. (2001) Nat. Genet. 28, 46-48]. Here we determine changes that occur in the mechanism of opening and closing of transmembrane channels formed by the GABA(A) receptor as a result of this mutation. The K289M mutation was introduced into the gamma2L subunit of the rat GABA(A) receptor, and the mutated subunit was coexpressed with the alpha1 and beta2 subunits in HEK293 cells. Transient kinetic techniques suitable for investigating reactions on cell surfaces with a microsecond-to-millisecond time resolution [Hess, G. P., and Grewer, C. (1998) Methods Enzymol. 291, 443-473] were used. They allow one to determine not only the channel-opening probability and rates of receptor desensitization but also the opening and closing rates of the mutated GABA(A) receptor channel. The channel-opening equilibrium constant of the mutated receptor was found to be 5-fold lower than that of the wild type. We calculated that this decrease in the channel-opening equilibrium accounts for the dysfunction of the mutated receptor. We discuss how a knowledge of the mechanism of the mutated receptor indicates an approach for alleviating this dysfunction.

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding.

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.

Structural Determinants of the Pharmacological Properties of the GABAAReceptor α6 Subunit

GABA(A) receptors are responsible for fast inhibitory neurotransmission in the mammalian brain and are the targets for many clinical drugs that act as antiepileptics, anxiolytics, and sedatives. The pharmacological characteristics of the receptor are largely determined by its subunit composition. Compared with all other alpha subtypes, the alpha6 subtype confers unique pharmacological properties. In particular, alpha6-containing receptors are more sensitive to both the agonist GABA and the antagonist amiloride. Results from chimeric constructs of the alpha1 and alpha6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for both these characteristics. Within this domain, we examined 15 amino acid residues unique to the alpha6 subtype. Each of these sites was individually mutated in the alpha6 subunit to the corresponding residue of the alpha1 subunit. The mutated subunits were expressed in human embryonic kidney-293T cells along with wild-type beta3 and gamma2L subunits and sensitivity to GABA and amiloride determined with whole-cell electrophysiological recordings. Serine83 in the alpha6 subunit influenced sensitivity to both GABA and amiloride. Leucine174 and tyrosine175 were also found to contribute to inhibition by amiloride but did not affect GABA sensitivity. These structural differences are at least partly responsible for the unique pharmacological properties associated with the alpha6 subunit.

An N-terminal histidine is the primary determinant of alpha subunit-dependent Cu2+ sensitivity of alphabeta3gamma2L GABA(A) receptors.

Copper (Cu2+) is a physiologically important cation and is released from nerve terminals. Cu2+ modulates GABAA receptor currents in an alpha subunit subtype-dependent manner; alpha1beta3gamma2L receptors are more sensitive to Cu2+ than alpha6beta3gamma2L receptors. We compared the effect of Cu2+ on alphabeta3gamma2L receptors containing each of the six alpha subtypes and generated alpha1/alpha6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for alpha subtype-dependent differences in Cu2+ sensitivity. Whole-cell GABAA receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant alpha subunits with beta3 and gamma2L subunits. Maximal Cu2+ inhibition of alpha1beta3gamma2L and alpha2beta3gamma2L receptor currents was larger (52.2 +/- 3.0 and 59.0 +/- 2.5%, respectively) than maximal inhibition of alpha3beta3gamma2L, alpha4beta3gamma2L, alpha5beta3gamma2L, and alpha6beta3gamma2L receptor currents (22.6 +/- 3.1, 19.2 +/- 3.4, 20.2 +/- 4.8, and 21.2 +/- 3.6%, respectively). Receptors containing chimeric constructs with alpha1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu2+ to an extent similar to those with alpha1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu2+ sensitivity. alpha1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the alpha2 subtype) conferred higher Cu2+ sensitivity, and the H141 residue was the major determinant in the motif. The beta3 subtype M2 domain residue H267, which is a major determinant of Zn2+ inhibition, and alpha6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn2+ sensitivity of the alpha6 subtype, also seemed to contribute to Cu2+ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in alpha1 and alpha2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu2+, that residue H141 is the major determinant in that motif, and that Cu2+ may also interact with GABAA receptors at sites similar to or overlapping Zn2+ sites.

Slow phases of GABAA receptor desensitization: structural determinants and possible relevance for synaptic function

GABA(A) receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA(A) receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing alpha1beta3delta receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABA(A) receptor activation.

The gamma subunit determines whether anesthetic-induced leftward shift is altered by a mutation at alpha1S270 in alpha1beta2gamma2L GABA(A) receptors.

A major action of volatile anesthetics is enhancement of gamma-aminobutyric acid receptor type A (GABA(A)R) currents. In recombinant GABA(A)Rs consisting of several subunit mixtures, mutating the alpha1 subunit serine at position 270 to isoleucine [alpha1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether alpha1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABA(A)Rs in mammalian brain are thought to consist of alpha1, beta2, and gamma2 subunits, and the alpha1(S270I) mutation has not been studied in the context of this combination. Recombinant GABA(A)Rs composed of alpha1beta2 or alpha1beta2gamma2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 microM to 1 mM) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes. In wild-type alpha1beta2gamma2L GABA(A), anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The alpha1(S270I) mutation itself rendered the GABA(A)R more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in alpha1(S270I)beta2gamma2L versus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in alpha1(S270I)beta2gammaL GABA(A)Rs. In parallel studies on alpha1beta2 (gamma-less) GABA(A)Rs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the alpha1(S270I) mutation nearly eliminated anesthetic-induced leftward shift. The results support a role for alpha1S270 in alpha1beta2gamma2L GABA(A)R gating and sensitivity to inhibition by volatile anesthetics. The alpha1S270 locus also modulates anesthetic enhancement in alpha1beta2 GABA(A)R. The presence of the gamma2L subunit reduces anesthetic-induced left shift of wild-type GABA(A)R and nullifies the impact of the alpha1(S2701) mutation on anesthetic modulation. Thus, the gamma2L subunit plays a significant role in GABA(A)R modulation by volatile anesthetic compounds.

Two gamma2L subunit domains confer low Zn2+ sensitivity to ternary GABA(A) receptors.

The sensitivity of GABAA receptors (GABARs) to Zn2+ inhibition depends on subunit composition. The predominant neuronal forms of mammalian GABARs, alpha(beta)gamma and, alpha(beta)delta are differentially sensitive to Zn2+ inhibition; alpha(beta)gamma receptors are substantially less sensitive than alpha(beta)delta receptors. Recently, functional domains involved in Zn2+ sensitivity have been identified in and subunits. Our aim in the present study was to localize functional domains of low Zn2+ sensitivity within gamma2L subunits. Chimeric subunits were constructed by progressively replacing the rat gamma2L subunit sequence with that of the rat delta subunit sequence. Whole-cell currents were recorded from mouse L929 fibroblasts coexpressing wild-type rat alpha1 and beta3 subunits with a chimeric delta-gamma2L subunit. Unlike alpha and beta subunits, the gamma2L subunit was found to contain a determinant of low Zn2+ sensitivity in the N-terminal extracellular region. In addition, we identified determinants in the M2 segment and the M2-M3 loop of the gamma2L subunit that are homologous to those found in beta and alpha subunits. We postulate that the interface between the latter two domains, which may form the outer vestibule of the channel, represents a single functional domain modulating Zn2+ sensitivity. Thus, the Zn2+ sensitivity of ternary GABARs appears to be determined by two functional domains, one in the N-terminal extracellular region and one near the outer mouth of the channel.

Effects of gamma-HCH and delta-HCH on human recombinant GABA(A) receptors: dependence on GABA(A) receptor subunit combination.

1. Human GABA(A) receptors containing different alpha and beta subunits with or without the gamma 2S or gamma 2L subunits were expressed in Xenopus oocytes and the effects of the insecticides gamma- and delta-hexachlorocyclohexane (gamma-HCH and delta-HCH, respectively) on these receptor subunit combinations were examined using two electrode voltage-clamp procedures. 2. gamma-HCH produced incomplete inhibition of GABA responses on all receptor combinations examined with affinities in the range of 1.1--1.9 microM. Affinity was not dependent on subunit composition but the maximum percentage of inhibition was significantly reduced in beta 1-containing receptors. delta-HCH both potentiated GABA(A) receptors and activated them in the absence of GABA at concentrations higher than those producing potentiation. Allosteric enhancement of GABA(A) receptor function by delta-HCH was not affected by the subunit composition of the receptor, By contrast the GABA mimetic actions of delta-HCH were abolished in receptors containing either alpha 4, beta 1 or gamma 2L subunits. 4. Sensitivity to the direct actions were not restored in receptors containing the mutant beta 1(S290N) subunit, but alpha 1 beta 2 gamma 2L receptors became sensitive to the direct actions of delta-HCH when oocytes were treated for 24 h with the protein kinase inhibitor isoquinolinesulphonyl-2-methyl piperazine dihydrochloride (H-7). 5. We have shown the influence of various alpha, beta and gamma subunits on the inhibitory, GABA mimetic and allosteric effects of HCH isomers. The data reveal that neither the inhibitory actions of gamma-HCH nor the allosteric effects delta-HCH has a strict subunit dependency. By contrast, sensitivity to the direct actions of delta-HCH are abolished in receptors containing alpha 4, beta 1 or gamma 2L subunits.

Semiquantitative analysis of the expression of GABA-A receptor subunits in the developing embryonic chick brain stem.

The expression levels of seven types of gamma-aminobutyric acid-A (GABA-A) receptor subunits (alpha1, beta2, beta3, beta4, gamma1, gamma2, and gamma4) were quantified in the embryonic chick brain stem at 2 to 20 d of incubation (E2 to E20) and just after hatching. The expression level of mRNA was measured by using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). When property regions were amplified, two products were observed for each of the beta2, beta4, and gamma2 subunits because of alternative splicing. These were named beta2S and beta2L, beta4S and beta4L, and gamma2S and gamma2L for shorter and longer fragments, respectively. Transcripts of alpha1, beta2L, beta2S, beta3, beta4L, beta4S, gamma1, and gamma2S subunits were first detected from E2 to E5 brain stems. The expression level of each subunit increased gradually with development and reached a plateau at E9 to E12. In contrast, a delay occurred in the appearance of both the gamma4 and gamma2L subunits, which were not detected until E8 to E10. The absence of gamma4 and/or gamma2L subunits may explain differences in the pharmacological characteristics of GABA-A receptors at the early stages of development.

Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

Behavioural changes produced by transgenic overexpression of gamma2L and gamma2S subunits of the GABAA receptor.

Transgenic mice overexpressing either the mouse gamma2L or gamma2S subunit of the GABAA receptor were generated in a C57BL/6 J x DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam- and ethanol-enhanced 36Cl- flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]-flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the gamma2L or gamma2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor gamma2 subunit available (regardless of whether it is gamma2L or gamma2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.

Dual action of isoflurane on the gamma-aminobutyric acid (GABA)-mediated currents through recombinant alpha(1)beta(2)gamma(2L)-GABA(A)-receptor channels.

Isoflurane (ISO) increased the agonist-induced chloride flux through the gamma-aminobutyric acid A receptor (GABA(A)R). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABA(A)R. We tested the hypothesis that, in addition to a blocking effect, ISO increases gamma-aminobutyric acid (GABA)-gated currents through recombinant GABA(A)R channels. HEK293 cells were transfected with rat cDNA for alpha(1),beta(2),gamma(2L) subunits. Currents elicited by 1 mM or 0. 01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABA(A)R. The balance between potentiation and block depends on the concentrations of both ISO and GABA.

Subunit mutations affect ethanol actions on GABAA receptors expressed in Xenopus oocytes

1. Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha2, beta1 and gamma2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2. Mutant alpha2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha2beta1gamma2L receptor. The mutation S265I (TM2) of beta1 and S280I (TM2) or S30IW (TM3) in gamma2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta1 subunit resulted in a receptor that was tonically open. 3. Using an EC5 concentration of GABA, the function of the wild-type receptor with alpha2beta1gamma2L subunits was potentiated by ethanol (50-200 mM). The mutations in TM2 or TM3 of the alpha2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha2 and beta1 TM2 mutants with a wild-type gamma2L subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma2L TM2 and/or TM3 mutants. 4. From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.

Hormone-dependent regulation of GABAA receptor gamma subunit mRNAs in sexually dimorphic regions of the rat brain.

Transmission mediated by gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) and the ventromedial nucleus (VMN) of the hypothalamus is known to play critical, but contrasting, roles in regulating steroid-dependent sexual behaviours in rats. Previous studies have demonstrated a striking dichotomy in receptor composition between the two regions with regard to gamma, but not alpha or beta, subunit expression. To test if gonadal steroids regulate the expression of the gamma subunit genes within the mPOA and the VMN, in situ hybridization analysis for messenger RNAs encoding the gamma 1, gamma 2Short (gamma 2S) and gamma 2Long (gamma 2L) subunits was done in gonadectomized male and female rats and in gonadally intact females over the oestrous cycle. No significant differences in the expression of the gamma subunit mRNAs were observed in gonadectomized male versus female rats. Significant effects of gonadal state in female rats were observed for gamma 1 mRNA levels in the mPOA and gamma 2L levels in the VMN. These data demonstrate that gonadal hormones exert activational control of expression of GABAA receptor gamma subunit mRNAs and suggest that differences in receptor structure may contribute to the functional modulation of female sexual behaviours mediated by GABAergic transmission in these regions.

Modulation of GABAA receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABAA receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABAA receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABAA receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABAA receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABAA receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABAA receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABAA receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABAA receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.Key words: GABAA receptor, neurosteroid, allopregnanolone, alphaxalone, Xenopus oocyte.

Molecular and functional adaptation of the GABA(A) receptor complex during pregnancy and after delivery in the rat brain.

The abundance of gamma-aminobutyric acid receptor type A (GABAA receptor) subunit mRNAs and polypeptides as well as muscimol-stimulated 36Cl- uptake were measured in rat cerebral cortex or hippocampus at various times during pregnancy and after delivery. RNase protection assays revealed that the amount of the gamma2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, and then returned to control values around the time of delivery. A similar pattern was observed for the alpha5 subunit mRNA in the cerebral cortex, whereas no significant changes were apparent for alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3 and gamma2S subunit mRNAs. The amounts of gamma2 and alpha1 proteins in the cerebral cortex were measured by immunoblot analysis; whereas the abundance of gamma2 protein decreased during pregnancy, no change was detected in the amount of alpha1 protein. Evaluation for functional significance of the down-regulated gamma2 and alpha5 subunit was made by determining the GABAA receptor function assessed by measurement of muscimol-stimulated 36Cl- uptake in cerebral cortical membrane vesicles. Muscimol-induced 36Cl- uptake was markedly reduced during of pregnancy compared with rats in oestrus. At this same time, the potentiating effects of diazepam and allopregnanolone on muscimol stimulation of 36Cl- uptake also were reduced. In contrast, the effects of muscimol, allopregnanolone and diazepam were significantly increased, relative to animals in oestrus, after delivery.