Identification of Transduction Elements for Benzodiazepine Modulation of the GABAAReceptor: 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 (χ), we demonstrated that although the N-terminal 167 γ₂ amino acid residues confer high-affinity BZD binding, other γ₂domains couple BZD binding to potentiation of the GABA-mediated Cl⁻ current (I_GABA). To determine which γ₂ regions couple binding to potentiation, we generated χs 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 ofI_GABA. Residues in γ₂199–236, γ₂224–236 (pre-M1), and particularly γ₂257–297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of I_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 γ₂257–297 domain revealed that three residues in concert, γ₂T281, γ₂I282 (M2 channel vestibule), and γ₂S291 (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.