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Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking
Published on: March 28, 2014
Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are
1Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
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 gamma(2)/alpha(1) chimeric subunits (chi), we demonstrated that although the N-terminal 167 gamma(2) amino acid residues confer high-affinity BZD binding, other gamma(2) domains couple BZD binding to potentiation of the GABA-mediated Cl(-) current (I(GABA)). To determine which gamma(2) regions couple binding to potentiation, we generated chis with longer N-terminal gamma(2) segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 gamma(2) residues showed incremental gains in maximal potentiation for diazepam enhancement of I(GABA). Residues in gamma(2)199-236, gamma(2)224-236 (pre-M1), and particularly gamma(2)257-297 (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, beta-carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the gamma(2)257-297 domain revealed that three residues in concert, gamma(2)T281, gamma(2)I282 (M2 channel vestibule), and gamma(2)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.
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