Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the ␥-aminobutyric acid type A receptor (GABA A -R). A conserved arginine residue in the TM2-TM3 loop of the GABA A -R ␣ 2 subunit was mutated to alanine, and the mutant ␣ 2 (R274A) was co-expressed with wild-type ␤ 1 and ␥ 2S subunits in human embryonic kidney (HEK) 293 cells. The GABA EC 50 was increased by about 27-fold in the mutant receptor relative to receptors containing a wildtype ␣ 2 subunit. Similarly, the GABA EC 50 at ␣ 2 (L277A)-␤ 1 ␥ 2S and ␣ 2 (K279A)␤ 1 ␥ 2S GABA A -R combinations was increased by 51-and 4-fold, respectively. The ␣ 2 (R274A) or ␣ 2 (L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA A -R. Based on these results, we propose that ␣ 2 (Arg-274) and ␣ 2 (Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA A -R.The GABA A 1 receptor (GABA A -R) as well as the glycine (Gly-R) and nicotinic acetylcholine receptors belong to a homologous gene family of ligand-gated ion channels (1, 2). The GABA A -R is made up of five glycoprotein subunits that have been proposed to contain four transmembrane domains (TM1-TM4) with part of the TM2 sequence of each subunit contributing to the channel pore (3, 4). The native GABA A -Rs are most commonly composed of two ␣ subunits, two ␤ subunits, and one ␥ subunit (5, 6). To date, six ␣-, four ␤-, and four ␥-subunit isoforms have been cloned in addition to a number of evolutionarily related subunit families (7,8).A diagram of the presumed topology of the GABA A -R ␣ 2 subunit (Fig. 1A) shows those residues in the N-terminus that have been implicated as possible agonist contact points. Residues Tyr-157, Thr-160, Thr-202, and Tyr-205 in the ␤ 2 subunit have also been proposed to contribute to the GABA binding site (9). The diagram displays the position of the ␣ 2 (Arg-274) residue that is the primary focus of this study and is located within the predicted short extracellular loop between TM2 and TM3. The function of the TM2-TM3 loop in GABA A -R subunits is currently unknown, but the corresponding region of the Gly-R ␣ subunit has been proposed to influence the efficiency of agonist-induced gating (10). A partial sequence alignment of Gly-R ␣ 1 compared with GABA A -R ␣ 2 , ␤ 1 , and ␥ 2 subunits (Fig. 1B) illustrates the similarity between the amino acid sequences in the TM2-TM3 loop within the group of receptor subunits. We therefore predicted that mutations in this region of the GABA A -R will produce similar effects on receptor gating.GABA A -R amino acid residues ␣ 2 (Arg-274), ␣ 2 (Leu-277), and ␣ 2 (Lys-279) are homologous to the residues in the Gly-R ␣ 1 subunit that alter the glycine EC 50 when mutated (Arg-271 (11), Leu-274 (12), and Lys-276 (13); Fig. 1B, boldface residues). ...

Human hereditary hyperekplexia ("startle disease") is a neurological disorder characterized by exaggerated, convulsive movements in response to unexpected stimuli. Molecular genetic studies have shown that this disease is often caused by amino acid substitutions at arginine 271 to glutamine or leucine of the ␣ 1 subunit of the inhibitory glycine receptor (GlyR). When exogenously expressed in Xenopus oocytes, agonist responses of mutant ␣ 1 (R271Q) and ␣ 1 (R271L) GlyRs show higher EC 50 values and lower maximal inducible responses (relative efficacies) compared with oocytes expressing wild-type ␣ 1 GlyR subunits. Here, we report that the maximal glycine-induced currents (I max ) of mutant ␣ 1 (R271Q) and ␣ 1 (R271L) GlyRs were dramatically potentiated in the presence of the anesthetic propofol (PRO), whereas the I max of wild-type ␣ 1 receptors was not affected. Quantitative analysis of the agonist responses of the isofunctionally substituted ␣ 1 (R271K) mutant GlyR revealed that saturating concentrations of PRO decreased the EC 50 values of both glycine and the partial agonist ␤-alanine by Ͼ10-fold, with relative efficacies increasing by 4-and 16-fold, respectively. Transgenic (tg) mice carrying the ␣ 1 (R271Q) mutation (tg271Q-300) have both spontaneous and induced tremor episodes that closely resemble the movements of startled hyperekplexic patients. After treatment with subanesthetic doses of PRO, the tg271Q-300 mutant mice showed temporary reflexive and locomotor improvements that made them indistinguishable from wild-type mice. Together, these results demonstrate that the functional and behavioral effects of hyperekplexia mutations can be effectively reversed by drugs that potentiate GlyR responses.

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We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

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