Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: Three residues are required for allosteric coupling

Abstract

Modulation of GABA(A) receptors by benzodiazepines (BZDs) is believed to involve two distinct steps: a recognition step in which BZDs bind and a conformational transition step in which the affinity of the receptor for GABA changes. Previously, using γ2/α1 chimeric subunits (X), we demonstrated that although the N-terminal 167 γ2 amino acid residues confer high- affinity BZD binding, other γ2 domains couple BZD binding to potentiation of the GABA-mediated Cl- current (/(GABA)). To determine which γ2 regions couple binding to potentiation, we generated X(s) with longer N-terminal γ2 segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 γ2 residues showed incremental gains in maximal potentiation for diazepam enhancement of/(GABA). Residues in γ2 199-236, γ2 224-236 (pre-M1), and particularly γ2 257-297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of/(GABA). In contrast, β-carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the γ2 257-297 domain revealed that three residues in concert, γ2 T281, γ2 1282 (M2 channel vestibule), and γ2 S291 (M2-M3 loop) are necessary to impart full BZD potentiation to chimeric receptors. Thus, these residues participate in coupling distant BZD-binding events to conformational changes in the GABA(A) receptor. The location of these novel residues provides insight into the mechanisms underlying allosteric coupling for other members of the ligand- gated ion channel superfamily.