Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor

Abstract

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]-strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr .apprx. 7300, and cyanogen bromide gave a labeled peptide of Mr 6200. Examination of the cleavage sites of these proteolytic reagents in the known primary sequence of the 48-kDa subunit [Grenningloh et al. (1987) Nature 328, 215-220] indicates that the [3H]strychnine binding site is located within amino acids 171-220 of this protein and probably involves tyrosine-197 or tyrosine-202. The proposed antagonist binding site corresponds to an extracellular domain close to the first transmembrane region of the 48-kDa subunit. A similar location of ligand binding sites has been reported for the .alpha.-subunits of nicotinic acetylcholine receptors. Thus, our results further reinforce the structural homologies observed between members of the ligand-gated receptor-channel family.