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The Journal of Neuroscience, December 1, 1999, 19(23):10213-10220

Identification of Transduction Elements for Benzodiazepine Modulation of the GABA_A Receptor: Three Residues Are Required for Allosteric Coupling

Andrew J. Boileau and Cynthia Czajkowski

Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706

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 ₂/₁ chimeric subunits (), we demonstrated that although the N-terminal 167 ₂ amino acid residues confer high-affinity BZD binding, other ₂ domains couple BZD binding to potentiation of the GABA-mediated Cl current (I_GABA). To determine which ₂ regions couple binding to potentiation, we generated s with longer N-terminal ₂ segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 ₂ residues showed incremental gains in maximal potentiation for diazepam enhancement of I_GABA. Residues in ₂199-236, ₂224-236 (pre-M1), and particularly ₂257-297 (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, -carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the ₂257-297 domain revealed that three residues in concert, ₂T281, ₂I282 (M2 channel vestibule), and ₂S291 (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.

Key words: GABA; GABA_A receptor; benzodiazepines; benzodiazepine-binding site; allosteric coupling; chimeric subunits; mutagenesis; inverse agonist; positive modulation; subunit; subunit; M2 domain; M2-M3 loop; Xenopus oocytes

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Copyright © 1999 Society for Neuroscience  0270-6474/99/192310213-08$05.00/0

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