The Major Site of Photoaffinity Labeling of the γ-Aminobutyric Acid Type A Receptor by [3H]Flunitrazepam Is Histidine 102 of the α Subunit

Abstract

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).

Mechanisms of neurosteroid interactions with GABAA receptors

“…Photoaffinity labeling has successfully been used to locate the benzodiazepine (Davies et al, 1996;Duncalfe et al, 1996;Sawyer et al, 2002;Wieland et al, 1992) and muscimol (Smith & Olsen, 1994) binding sites on the GABA A receptor, and sites for etomidate (Husain et al, 2006;Ziebell et al, 2004) and octanol (Pratt et al, 2000) on the Torpedo nicotinic acetylcholine receptor. In principle photoaffinity labeling techniques should provide a comprehensive search of all possible protein targets and should not be confounded by either multiple regions of protein contributing to a binding site or by the existence of multiple binding sites.…”

Low dose acute alcohol effects on GABAA receptor subtypes

“…Consistent with this view are 2 recent reports of behavioral EtOH antagonist activity of Ro15-4513/flumazenil congeners named RY024 and RY023, that have a cyano-or an acetylene -instead of an azido -group at the C7 position of the BZ ring (McKay et al, 2004;Cook et al, 2005). In addition, it has been shown that the C7 position in classical benzodiazepines is close to the a critical histidine residue in GABA A R α1,2,3,5 subunits (α1-H101) (Berezhnoy et al, 2004) that is required for sensitivity of GABA A R to classical benzodiazepines in vitro (Wieland et al, 1992;Benson et al, 1998) and in vivo (Rudolph et al, 1999) and photolabeled by [ 3 H]flunitrazepam (Duncalfe et al, 1996). The residue homologous to this histidine is an arginine (R100) in α4 and α6 GABA A R subunits, and as described above in Section 2.5, leads to increased EtOH sensitivity of β3 and δ subunitcontaining receptors in vivo and in vitro .…”

Two neighboring residues of loop A of the α1 subunit point towards the benzodiazepine binding site of GABAA receptors

“…Mutagenesis studies identified the cleft between a and c subunits as the binding pocket for benzodiazepines [12][13][14]. a 1 H101 was identified as the target of photoaffinity labeling by [ 3 H] flunitrazepam [15] and a 1 Y209 as the target of [ 3 H] Ro15-4513 [16]. Pharmacophore modeling attempted to describe the shape of the binding pocket [17,18].…”