Deduction of amino acid residues in the GABA_A receptor α subunits photoaffinity labeled with the benzodiazepine flunitrazepam

Abstract

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acid_A receptor modulator [³H]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74–123 of the bovine α1 subunit, in agreement with a previously predicted labeling domain between residues 59–148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of ³H counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of ³H counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [³H]flunitrazepam is His102 of the bovine α1 subunit. In addition we also detected possible secondary labeling of Pro97.

Introduction

Benzodiazepines (BZ) are clinically important central nervous system depressants with anxiolytic, sedative, antiepileptic and muscle relaxant therapeutic action (Olsen and Venter, 1986). They act by allosteric modulation of the inhibitory action of γ-aminobutyric acid (GABA) on GABA_A receptor channels, binding to a site separate from the GABA agonist site, and increasing the frequency of GABA-mediated channel opening (Olsen, 1981).

The α subunit of the GABA_A/benzodiazepine receptor complex is thought to be the major determinant for BZ pharmacology (Olsen and Tobin, 1990) and the 51 kDa α subunit is the primary site for photoaffinity labeling by the nitrobenzodiazepine agonist [³H]flunitrazepam ([³H]FLU) (Möhler et al., 1980, Sieghart and Karobath, 1980, Sigel et al., 1983, Stauber et al., 1987.) This polypeptide has been identified as the α1 gene product using subtype-specific antisera (Duggan and Stephenson, 1990, Stephenson et al., 1990, Endo and Olsen, 1993) and is abundant in mammalian cortex, as well as being the only BZ agonist-binding GABA_A subunit found in the cerebellum (Wisden et al., 1988). The predicted polypeptide topology has an extracellular domain, comprising about half the mass, and four putative transmembrane spanning domains connected by loops of varying lengths. The α1 subunit clone sequence contains two consensus sites for N-linked glycosylation in the extracellular domain (Schofield et al., 1987) and [³H]FLU-labeled polypeptides have been shown to be glycosylated (Sieghart and Fuchs, 1988). Although the γ subunit appears to be required for expression of BZ binding and BZ enhancement of GABA-mediated channel function in transfected cells (Pritchett et al., 1989a), photolabeling of this subunit with [³H]FLU is less than that of α, possibly nil, as determined by immunoprecipitation of denatured receptor with α1- and γ2-specific antibodies (Stephenson et al., 1990). Fluorography of SDS-PAGE-separated [³H]FLU-photolabeled polypeptides shows multiple higher molecular weight bands which vary with brain region (Sieghart and Karobath, 1980) and ligand binding affinity (Sieghart et al., 1983) corresponding to other α subunits (Bureau and Olsen, 1988, Fuchs et al., 1988, Huh et al., 1996).

Previous evidence indicated that the domain for BZ photolabeling in cultured chick cells resides in a 24 kDa tryptic fragment which remains associated with the plasma membrane following proteolytic treatment of intact cells; receptor treated in this manner retains the ability to bind [³H]FLU both reversibly and irreversibly (Czajkowski and Farb, 1986). Trypsinization of photolabeled rat brain membrane homogenates also results in a labeled membrane-bound fragment of ca. 28 kDa (Klotz et al., 1984). In a subsequent study (Stephenson and Duggan, 1989), photolabeled purified bovine receptor was denatured and digested with cyanogen bromide (CNBr), and the fragments separated by SDS-PAGE and identified by immunoblotting. A single 11 kDa labeled fragment was detected and this was not recognized by any of three N- and C-terminal sequence-specific antibodies. The authors concluded, based upon the predicted molecular weight of peptides theoretically generated from a total CNBr digest of the bovine α1 subunit, that the site for the photoaffinity reaction must reside between residues 59–148, in the putative extracellular domain. More recently, Duncalfe et al. (1996) used the release of radioactivity from Edman degradation of a purified [³H]FLU labeled CNBr fragment to identify the major site of photoaffinity labeling as His102 of the bovine α1 subunit. In the present study, we have examined the [³H]FLU photoaffinity substrate site by peptide mapping and microsequencing of partially purified photolabeled peptides and conclude that the primary substrate for the photoaffinity reaction in the bovine α1 subunit is extracellular, corresponding to His102, with secondary labeling of Pro97.