Barbiturates require the N terminus and first transmembrane domain of the delta subunit for enhancement of alpha1beta3delta GABAA receptor currents

Abstract

GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.

FIGURE 1.

Representative GABA current traces prior to and after pentobarbital modulation for wild type and δ-γ2L chimeric GABA_A receptors. A, amino acid alignment for M1 domain of γ2L and δ subunits. δ-γ2L chimera splice sites were indicated by dashed lines. B–F, representative whole cell current traces evoked by a saturating concentration of GABA (1 mm) (left traces) as well as co-application of GABA (1 mm) and pentobarbital (100 μm) with pentobarbital (PB, 100 μm) preapplied for 1.5 s (right traces). The GABA control currents (gray traces) were normalized to the currents evoked by co-application of GABA and pentobarbital to show the alterations of desensitization and deactivation. A schematic of GABA_A receptor wild type subunit γ2L (white shading), δ (gray shading), or a chimeric subunit is depicted before each set of traces. N represents the N terminus of the subunit, and numbers 1 to 4 denote M1–M4 of the subunit. The solid line above each current trace denotes the duration (4 s) of GABA application, and the hatched bar represents the duration of pentobarbital application. The horizontal time scale of C, D, E, and F is the same as that of B.

FIGURE 2.

Pentobarbital modulation of peak currents, desensitization, and deactivation of wild type and δ-γ2L chimeric receptors. A, shown is the mean extent of enhancement by pentobarbital (PB) of wild type and δ-γ2L chimeric receptors. The dashed line indicates 100%. B, shown is a comparison of the mean desensitization prior to and after pentobarbital treatment among wild type and δ-γ2L chimeric receptors. C, the mean deactivation time constant was greater after pentobarbital treatment as compared with GABA control for wild type and δ-γ2L chimeric receptors. ++, significantly different from γ2L, M1e, or M1pre-iso at p < 0.01; +++, p < 0.001 (one-way analysis of variance followed by Newman-Keuls multiple comparison test); *, significantly different from GABA control at p < 0.05; **, p < 0.01; ***, p < 0.001 (paired Student's t test).

FIGURE 3.

Representative GABA current traces prior to and after pentobarbital modulation for wild type and γ2L-δ chimeric receptors. A, amino acid alignment for M1 domain of δ and γ2L subunits. γ2L-δ chimera splice sites were indicated by dashed lines. B–F, representative whole cell current traces evoked by a saturating concentration of GABA (1 mm) (left traces) as well as co-application of GABA (1 mm) and pentobarbital (PB, 100 μm) with pentobarbital (100 μm) preapplied for 1.5 s (right traces). The GABA control currents (gray traces) were normalized to the currents evoked by co-application of GABA and pentobarbital to show the alterations of desensitization and deactivation. A schematic of the GABA_A receptor wild type subunit δ (gray shading), γ2L (white shading), or chimeric subunit was shown before each set of traces. N represents N terminus of the subunit, and numbers 1–4 denote M1–M4 of the subunit. The solid line above each current trace denotes the duration (4 s) of GABA application, and the hatched bar represents the duration of pentobarbital application. The horizontal time scale of C, D, E, and F is the same as that of B.

FIGURE 4.

Pentobarbital modulation of peak currents, desensitization and deactivation of wild type and γ2L-δ chimeric receptors. A, shown is the mean extent of enhancement by pentobarbital (PB) of wild type and γ2L-δ chimeric receptors. The dashed line indicates 100%. B, shown is the comparison of the mean desensitization prior to and after pentobarbital treatment among wild type and γ2L-δ chimeric receptors. C, the mean deactivation time constant was greater after pentobarbital treatment as compared with GABA control for wild type and γ2L-δ chimeric receptors. +++, significantly different from M1e′, M1p′, M1i′, and γ2L at p < 0.001 (one-way analysis of variance followed by Newman-Keuls multiple comparison test); *, significantly different from GABA control at p < 0.05; **, p < 0.01; ***, p < 0.001 (paired Student's t test).