Structural Determinants of Benzodiazepine Allosteric Regulation of GABA_AReceptor Currents

Abstract

Benzodiazepine enhancement of GABA A receptor current requires a ␥ subunit, and replacement of the ␥ subunit by the ␦ subunit abolishes benzodiazepine enhancement. Although it has been demonstrated that benzodiazepines bind to GABA A receptors at the junction between ␣ and ␥ subunits, the structural basis for the coupling of benzodiazepine binding to allosteric enhancement of the GABA A receptor current is unclear. To determine the structural basis for this coupling, the present study used a chimera strategy, using ␥2L-␦ GABA A receptor subunit chimeras coexpressed with ␣1 and ␤3 subunits in human embryonic kidney 293T cells. Different domains of the ␥2L subunit were replaced by ␦ subunit sequence, and diazepam sensitivity was determined. Chimeric subunits revealed two areas of interest: domain 1 in transmembrane domain 1 (M1) and domain 2 in the C-terminal portion of transmembrane domain 2 (M2) and the M2-M3 extracellular loop. In those domains, site-directed mutagenesis demonstrated that the following two groups of residues were involved in benzodiazepine transduction of current enhancement: residues Y235, F236, T237 in M1; and S280, T281, I282 in M2 as well as the entire M2-M3 loop. These results suggest that a pocket of residues may transduce benzodiazepine binding to increased gating. Benzodiazepine transduction involves a group of residues that connects the N terminus and M1, and another group of residues that may facilitate an interaction between the N terminus and the M2 and M2-M3 loop domains.

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

“…It is possible that L293 is a key residue in regions linking the allosteric site of 5-HI binding to agonist binding and/or channel gating. In this regard, it is interesting to note that amino acid residues in the TM2 domain and TM2-TM3 loop of the GABA A receptor are the structural determinants for coupling benzodiazepine binding to increased channel gating (Boileau & Czajkowski, 1999;Jones-Davis et al, 2005).…”

“…It has been established that amino acid residues in the transmembrane (TM) 2 domain and TM2-TM3 loop play critical roles in 5-HT 3 receptor gating (Filatov & White, 1995; and allosteric modulation (Boileau & Czajkowski, 1999;Hu & Lovinger, 2005;Jones-Davis et al, 2005) of the Cys-loop ligand-gated ion channels. To identify amino acid residue(s) that participate in 5-HI modulation, we examined the roles of selected residues from TM2 domain of the 5-HT 3A receptor by site-directed mutagenesis and whole-cell patch-clamp recording.…”

Identification of Structures within GABAAReceptor α Subunits That Regulate the Agonist Action of Pentobarbital

“…The observation that multiple residues or structural domains combine to produce the distinctive pharmacological or functional properties associated with a GABAR subunit is not uncommon. For example, a complex interaction of several residues underlies the differing sensitivities of the ␦ and ␥2 subunits to benzodiazepine agonists (Jones-Davis et al, 2005), the ␣ subtypes to GABA (Böhme et al, 2004), and the ␣6 subtype to inhibition by amiloride (Drafts and Fisher, 2004). Identification of all of the structural components that contribute to the action of pentobarbital will probably require study of multiple chimeric constructs that exchange smaller segments of the extracellular N-terminal domains of these subunits.…”

Alterations of GABAA-Receptor Function and Allosteric Modulation During Development of Status Epilepticus

“…GABA A receptors are allosterically modulated in a subunit-dependent manner by a variety of structurally different positive and negative modulators, including benzodiazepines, barbiturates, neurosteroids, penicillin, and zinc (Olsen and Macdonald 2002). For example, benzodiazepines are positive modulators for ␥2 subunitcontaining receptors and multiple domains of the ␥2 subunit are involved in their allosteric modulation (Boileau and Czajkowski 1999;Jones-Davis et al 2005). In contrast, zinc is a negative modulator for ␥2-subunit-containing receptors, whose action may involve overlapping ␥2 subunit domains with those for benzodiazepines (Nagaya and Macdonald 2001).…”