Identification of an Amino Acid Defining the Distinct Properties of Murine β₁ and β₃ Subunit‐Containing GABA_A Receptors

Abstract

Murine ␥-aminobutyric acid (GABA) type A homomeric receptors made of ␤ 1 subunits are profoundly different, when expressed in Xenopus oocytes, from ␤ 3 homomeric receptors. Application of the intravenous general anesthetic pentobarbital, etomidate, or propofol to ␤ 3 homomeric receptors allows current flow. In contrast, ␤ 1 homomers do not respond to any of these agents. Through construction of chimeric ␤ 1 /␤ 3 receptors, we identified a single amino acid that determines the pharmacological difference between the two ␤ subunits. When the serine residue present in the wild-type nonresponsive ␤ 1 subunit is replaced by an asparagine found in the same position in the ␤ 3 subunit, the resulting pointmutated ␤ 1 S265N forms receptors responsive to intravenous general anesthetics, like the wild-type ␤ 3 subunits. Conversely, after mutation of the wild-type ␤ 3 to ␤ 3 N265S, the homomeric receptor loses its ability to respond to these same general anesthetics. Wild-typeto-mutant titration experiments showed that the nonresponsive phenotype is dominant: A single nonresponsive residue within a pentameric receptor is sufficient to render the receptor nonresponsive. In ␣ 1 ␤ x or ␣ 1 ␤ x ␥ 2 heteromeric receptors, the same residue manifests as a partial determinant of the degree of potentiation of the GABA-induced current by some general anesthetics. The location of this amino acid at the extracellular end of the second transmembrane segment, its influence in both homomeric and heteromeric receptor function, and its dominant behavior suggest that this residue of the ␤ subunit is involved in an allosteric modulation of the receptor. Key Words: GABA A receptor-␤ subunit-Gating-Chimera-Site-directed mutagenesis-Intravenous general anesthetics. J. Neurochem. 74, 827-838 (2000).␥-Aminobutyric acid (GABA) type A (GABA A ) receptor is a member of the ligand-gated ion channel superfamily of homologous receptors (Schofield et al., 1987). Binding of the ligand, GABA, to the receptor opens the integral Cl Ϫ channel, driving the membrane potential toward the chloride equilibrium potential, thus reducing the sensitivity of the membrane to the effects of excitatory neurotransmitters. GABA and competitive GABA agonists such as muscimol, as well as antagonists such as bicuculline, bind to the extracellular N-terminal region of both the ␣ subunit (Sigel et al., 1992) and the ␤ subunit (Amin and Weiss, 1993). Similarly, benzodiazepines bind to the N-terminal region of the ␥ subunit (Mihic et al., 1994). General anesthetic drugs of diverse chemical structures, e.g., pentobarbital, propofol, etomidate, and volatile agents such as halothane, directly open the GABA A Cl Ϫ channel in the absence of GABA (Franks and Lieb, 1994). The site on the receptor where these drugs act has not been identified, but recent evidence suggests a critical role of the second transmembrane segment (M2) in the action of volatile general anesthetics, alcohol, and etomidate on GABA A receptors Mihic et al., 1997;Moody et al., 1997).The GABA A receptor is us...

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

“…An alternative explanation is that it may be part of a pivotal signal transduction point through which the effects of many of these drugs converge (13). Indeed, conformational changes corresponding to different functional states are greatly influenced by mutations within TMII (13,31,(42)(43). In the present experiments, the substitution of glutamine or asparagine for the native serine may have influenced a state transition that altered picrotoxin inhibition of the channel, resulting in the use-facilitated blockade.…”

Multiple Modes for Conferring Surface Expression of Homomeric β1 GABAA Receptors

“…The ␤1 subunits of various species have been shown to express in Xenopus oocytes as homo-oligomers (21,(33)(34)(35). However, ␤1 expression in somatic cell lines has been more variable, and any reported expression has been low (15,22,33,36).…”

“…In contrast, structural requirements for assembly of ␤1 homomeric receptors have not been examined. A small body of literature exists, describing expression and function of ␤1 homomeric receptors in Xenopus oocytes and somatic cell lines (21,22). This literature, however, shows unclear and often contradictory results depending on expression system and species of subunit used.…”

Differential Effects of Methylmercury on γ-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

“…For instance, the presence of the ␦ subunit increases the affinity of ␣ 6 -containing receptors for GABA and imparts sensitivity to inhibition by Zn 2ϩ (Saxena and Macdonald, 1994). Similarly, sensitivity of the GABA receptor to barbiturates depends on the identity of the ␤ subunit; inclusion of the ␤ 3 subunit makes the receptor insensitive to the effects of pentobarbital (Cestari et al, 2000). Furosemide inhibition is also enhanced when the ␤ 1 subunit of the GABA A receptor is replaced with either the ␤ 2 or ␤ 3 subunit subtypes (Thompson et al, 1999).…”

“…GABA A receptors containing the ␣ 6 or ␣ 1 subunit have unique pharmacological and biophysical properties, including differential sensitivity to agonists, such as benzodiazepines (Lü ddens and Wisden, 1991;Sieghart, 1992;Makela et al, 1997) and barbiturates (Fisher et al, 1997;Cestari et al, 2000) and to antagonists such as Zn 2ϩ (Draguhn et al, 1990;Saxena andMacdonald, 1994, 1996;Zempel and Steinbach, 1995), furosemide (Korpi et al, 1995), and La 3ϩ (Saxena et al, 1997;Makela et al, 1999). As such, expression of distinct subunits can alter significantly the pharmacological sensitivity of the receptor.…”