Type A yaminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligandgated ion channels probably formed from the coassembly of different subunits (a1.6, Pl39 8) in the arrangement apy or ad8. The activation of these receptors by GABA can be modulated by a range ofcompounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the P subunit of the GABAA receptor. The action of loreclezole depends on the type of 13 subunit present in the receptor complex; receptors containing P2 or 13 subunits have >300-fold higher affinity for loreclezole than receptors containing a PI subunit. We have used this property to identify the amino acid residue in the P subunit that determines the subunit selectivity of loreclezole. Chimeric P1/12 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human a, and y subunits. The chimera 131/P2Lys237-Gly334 conferred sensitivity to 1 IAM loreclezole. Within this region there are four amino acids that are conserved in P2 and 13 but differ in 1. By mutating single amino acids of the P1 subunit to the P2/13 equivalent, only the P1 mutation of Ser-290 -* Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the 12 and P3 subunits to the Pi equivalent (Asn --Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors coning the mutated 13 subunits. Thus, a single amino acid, 12 Asn-289 (P1 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.The mammalian type A )-aminobutyric acid (GABAA) receptor gene family is now known to consist of a number of subunit polypeptides (al-a6, (31-P3, 3v-y3. 6, pi-p2) (1, 2).There is an increased body of evidence that suggests that in neurons these subunits coassemble in the arrangement a(yor a(3S (3,4), probably as pentamers (by analogy with the related nicotinic receptor), to give a family of receptor subtypes that have different spatial and temporal patterns ofexpression (5).The activity of GABAA receptors can be allosterically modulated by a number of agents, including ethanol and neurosteroids, and the clinically important barbiturates and benzodiazepines (BZs) (6, 7). The BZ pharmacology of GABAA receptor subtypes has been studied in some detail.Only receptors made up of an a, 3, and 'y subunit exhibit high-affinity BZ binding (8), and the pharmacology is determined by the type of a (9-11) and y (12), but not the (, (13), subunit present, suggesting that this modulatory site is constituted by determinants on both the a and y subunits. The structural determinants that contribute to the other modulatory sites of the GABAA receptor are currently unclear.Loreclezole, {(Z)-1-[2-chloro-2-(2,4-dichlorophenyl) ethenyl]-1,2,4-triazole}, is a broad-spectrum anti...

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SUMMARYPharmacological analyses of ␥-aminobutyric acid A (GABA A ) receptor subtypes have suggested that both the ␣ and ␥ subunits, but not the ␤ subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different ␥ subunits in the receptor macromolecule to identify amino acids ␥2Phe77 and ␥2Met130 as key determinants of the benzodiazepine binding site. ␥2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-␤-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, ␥2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218,872, or methyl-␤-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of ␥1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that ␥2Phe77 and ␥2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the ␣ subunit, we demonstrated a critical role for the ␥ subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, ␥2Phe77 is homologous to ␣1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABA A receptor.The GABA A receptor, a member of the ligand-gated ion channel family, mediates synaptic inhibition through the gating of chloride ions, resulting in hyperpolarization of the cell membrane. It is the site of action of a number of pharmacological agents, including BZs, barbiturates, and anesthetics. The hetero-oligomeric receptor is formed from the coassembly of five different subunit classes [␣, ␤, ␥, ␦ (1, 2), and ⑀ (3, 4)] in a presumed pentameric arrangement (5, 6) to yield a family of receptor subtypes. It is the heterogeneity within these subunits that provides the molecular basis for the differences in pharmacology of receptor subtypes (7).Classic BZ pharmacology is exhibited by receptors containing a ␥2 subunit in combination with an ␣ and a ␤ subunit (8). The affinity of BZ ligands for the receptor is dependent on the ␣ subunit isoform, and hence compounds such as CL218,872 and zolpidem have higher affinity for ␣1␤n␥2 (n ϭ 1, 2, or 3) receptors than for other ␣ subunit-containing receptors (9, 10), and flunitrazepam and diazepam (11, 12) have very low affinity (Ͼ10 M) for ␣4␤n␥2 and ␣6␤n␥2. Mutagenesis studies have identified two amino acids on the ␣ subunit as contributing to the BZ binding site (13,14 McKernan et al. (16) that the ␥ subunit also contributes significantly to the BZ binding site. I...

1 We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA A b2 and investigated the actions of a number of GABA A agonists, antagonists and modulators on human a1b2DL259Sg2s compared to wild type a1b2g2s GABA A receptors, expressed in Xenopus oocytes. 2 The mutation resulted in smaller maximum currents to g-aminobutyric acid (GABA) compared to a1b2g2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation signi®cantly decreased the GABA EC 50 (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S) also displayed a decrease in EC 50 . 3 In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on a1b2DL259Sg2s receptors, with di erent degrees of maximum inhibition. 4 The e ects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC 20 ). Compared to wild type, none of these modulators potentiated the EC 20 response of a1b2DL259Sg2s receptors, however they all directly activated the receptor in the absence of GABA. 5 To conclude, the above mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site.

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with ␥-aminobutyric acid (GABA) A receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABA A receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABA A receptors expressed in Xenopus laevis oocytes. Replacement of the ␥2s subunit within the ␣1␤3␥2s receptor with the ⑀ subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The ␥2s subunit was not critical for potentiation because ␣1␤3 receptors were also potentiated by tracazolate. ␥2/⑀ chimeras revealed a critical Nterminal domain between amino acids 206 and 230 of ␥2, governing the nature of this response. Replacement of the ␤3 subunit with the ␤1 subunit within ␣1␤3␥2s and ␣1␤3⑀ receptors also revealed selectivity of tracazolate for ␤3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of ␥2s with ␥1 or ␥3 revealed a profile intermediate to that of ␣1␤1⑀ and ␣1␤1␥2s. ␣1␤1␦ receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC 20 was much greater than on ␣1␤1␥2. Concentration-response curves to GABA in the presence of tracazolate for ␣1␤1⑀ and ␣1␤1␥2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC 50 only on ␣1␤1␥2. Like ␣1␤1␥2s, GABA concentration-response curves on ␣1␤1␦ receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABA A receptors: its potency (EC 50 ) is influenced by the nature of the ␤ subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (␥1-3, ␦, or ⑀) within the receptor complex.

L-655,708 is a ligand for the benzodiazepine site of the g-aminobutyric acid type A (GABA A ) receptor that exhibits a 100-fold higher af®nity for a5-containing receptors compared with a1-containing receptors. Molecular biology approaches have been used to determine which residues in the a5 subunit are responsible for this selectivity. Two amino acids have been identi®ed, a5Thr208 and a5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher af®nity of this ligand at a5-containing receptors. L-655,708 is a partial inverse agonist at the GABA A receptor which exhibited no functional selectivity between a1-and a5-containing receptors and showed no change in ef®cacy at receptors containing a1 subunits where amino acids at both of the sites had been altered to their a5 counterparts (a1DSer205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also in¯uence the binding af®nities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA A receptor, and further delineate a region just N-terminal to the ®rst transmembrane domain of the receptor a subunit that contributes to this binding site. The mammalian g-aminobutyric acid type A (GABA A ) receptor is a pentameric structure containing different combinations of a1±6, b1±3, g1±3, d, 1 and u subunits (for reviews, see Barnard et al. 1998;Mehta and Ticku 1999). Among the many classes of drug which interact with the receptor are the benzodiazepines (BZs) that are used as anxiolytics, anticonvulsants and hypnotics. Immunoprecipitation with subunit speci®c antibodies, and recombinant receptor studies have demonstrated that high af®nity BZ-binding sites are found on receptors of abg 2 composition. The differential af®nity of drugs such as diazepam for such receptors in which the a1 subunit has been substituted by a6, demonstrating that the a subunit is a major contributor to the BZ binding site present on the receptor (Lu Èddens et al. 1990). Site-directed mutagenesis studies have been shown in a number of cases (e.g. Pritchett and Seeburg 1991;Wieland et al. 1992;Amin et al. 1997;Buhr et al. 1997) to identify residues within the a subunit which are critical for the drug±receptor interaction. Although the diazepam insensitivity of receptors containing a4 and a6 is well documented (e.g. Lu Èddens et al. 1990;Wisden et al. 1991), few clinically ef®cacious drugs other than zolpidem (which is 10-fold selective for a1-containing receptors over a2-and a3-containing receptors, with negligible af®nity for a4-, a5-and a6-containing receptors) discriminate between receptors containing other a subunits. Recently, however, the identi®cation of a ligand (L-655,708) with selectivity for the a5-containing receptor has been reported (Quirk et al.

GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other alpha-subunits. Furosemide exhibits approximately 100-fold selectivity for alpha6-containing receptors over alpha1-containing receptors. By making alpha1/alpha6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha6beta3gamma2s receptors. Within the alpha1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta-subunit selectivity of furosemide to be due to asparagine 265 in the beta2 and beta3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at alpha6beta3gamma2s compared with alpha1beta3gamma2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.

Transfection of mouse L(tk‐) cells with human N‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone‐inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l‐(ε)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4‐fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A‐I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.