Structural domains of the human GABAA receptor β3 subunit involved in the actions of pentobarbital

This study was conducted to search for the residues of the β3 subunit which affect pentobarbital action on the γ-aminobutyric acid type A (GABAA) receptor. Three chimeras were constructed by joining the GABAA receptor β3 subunit to the ρ1 subunit. For each chimera, the N-terminal sequence was derived from the β3 subunit and the C-terminal sequence from the ρ1 subunit, with junctions located between the membrane-spanning regions M2 and M3, in the middle of M2, or in M1, respectively. In receptors obtained by the coexpression of α1 with the chimeric subunits, in contrast with those obtained by the coexpression of α1 and β3, pentobarbital exhibited lower potentiation of GABA-evoked responses, and in the direct gating of Cl− currents, an increase in the EC50 together with a marked decrease in the relative maximal efficacy compared with that of GABA. Estimates of the channel opening probability through variance analysis and single-channel recordings of one chimeric subunit showed that the reduced relative efficacy for gating largely resulted from an increase in gating by GABA, with little change in efficacy of pentobarbital. A fit of the time course of the response by the predictions of a class of reaction schemes is consistent with the conclusion that the change in the concentration dependence of activation by pentobarbital is due to a change in pentobarbital affinity for the receptor. Therefore, the data suggest that residues of the β3 subunit involved in pentobarbital binding to GABAA receptors are located downstream from the middle of the M2 region. Pentobarbital affects GABAA receptor-mediated responses in several ways. At low micromolar concentrations it potentiates GABA-evoked responses, at high micromolar concentrations it opens GABAA receptors directly and at millimolar concentrations it reduces the response (Akaike et al. 1987b). Since the ability to enhance the ion channel activation of GABAA receptors is a common feature of several general anaesthetics (Franks & Lieb, 1994), the action on the GABAA receptor is a probable major molecular mechanism for anaesthetic action in the mammalian central nervous system (Tanelian et al. 1993). However, general anaesthetics do not exert the same effects on all the GABA-gated receptor Cl− ion channels (Thomson et al. 1996). In particular, those composed of ρ1 homomers exhibit little if any response to anaesthetics (Shimada et al. 1992). A combination of molecular biological, pharmacological and physiological approaches has provided a great deal of information on the portions of the GABAA receptor subunits which affect the response of benzodiazepines (see Smith & Olsen, 1995), of GABA (Amin & Weiss, 1993) and some classes of anaesthetics (Mihic et al. 1997). However, only a few recent studies (Birnir et al. 1997; Krasowski et al. 1998b; Amin, 1999) have provided some initial insights into the regions involved in the physiological effects of barbiturates. In the present study, chimeric subunits were generated between the human β3 subunit of the GABAA receptor and the human ρ1 subunit, with the goal of localizing amino acid residues affecting pentobarbital actions on GABAA receptor channels. Three constructs were prepared in which the upstream, N-terminal, part of the β3 subunit was joined to the downstream, C-terminal, part of ρ1 (Fig. 1). In each chimera, the junction was located in the region between the N-terminal end of M1 and the N-terminal end of M3. The junction was progressively moved upstream from the middle of the M2-M3 linker (c7), to the middle of M2 (c1), or M1 (c2). If the amino acids of β3 required for pentobarbital responses are replaced by the corresponding residues of ρ1, a decrease in activity should result. Studies on the residues involved in the binding of GABA to GABAA receptors (Amin & Weiss, 1993) and acetylcholine to nicotinic receptors (reviewed in Karlin & Akabas, 1995) indicate that amino acids responsible for the binding of a ligand may be located in several subunits (reviewed in Karlin & Akabas, 1995) and that within each subunit they can be distributed in widely separated regions (Amin & Weiss, 1993). Therefore, pentobarbital effects may not be necessarily eliminated by replacement of the binding residues of one subunit, and in each subunit they may be altered by mutations of residues lying in a long stretch of the subunit primary sequence. Figure 1 Summary of the chimeric subunits studied Because the hypnotic properties of barbiturates are related to lipid solubility (reviewed in Gallagher & Freer, 1985), binding sites for anaesthetics may be located in a lipophilic pocket of the receptor, and the transmembrane domains may possibly contain residues that bind pentobarbital. However, in the nicotinic acetylcholine receptor, these regions contain residues which can dramatically affect gating mechanisms, as well (reviewed in Karlin & Akabas, 1995). Therefore, to interpret observations we have also attempted to distinguish changes in affinity from changes in pentobarbital efficacy. Chimeric subunits were expressed in combination with GABAAα1 subunits, and initially concentration-effect curves were obtained for gating by GABA, for pentobarbital potentiation of GABA-evoked responses, for direct gating by pentobarbital and for block by pentobarbital. Then the relative maximal responses elicited by GABA and pentobarbital were compared and the Popen of channel activation by GABA and pentobarbital were estimated. Finally, the time courses of responses to pentobarbital were fitted by the predictions of a class of reaction schemes, to test the adequacy of our analysis. The results indicate that residues affecting pentobarbital potentiation of the GABA-evoked response are localized in a region of the β3 subunit extending from M1 to the M2-M3 linking region. Furthermore, in one chimera (c1, formed in the M2 region), the affinity of pentobarbital for the site involved in direct gating has been reduced. This suggests that residues involved in the binding of pentobarbital are located downstream of the middle of the M2 domain. The results of the study have been presented in preliminary form (Serafini et al. 1997, 1998).