The glycine receptor of rat spinal cord is an oligomeric membrane glycoprotein of molecular mass 250,000 daltons that contains three polypeptides of 48,000, 58,000, and 93,000 daltons. Monoclonal antibodies (mAbs) were prepared against the affinity-purified glycine receptor protein by using '5I-labeled receptor preparations for the detection of positive hybrids. From nine monoclonal antibodies obtained, six recognized denatured receptor polypeptides blotted to nitrocellulose paper. Two of these antibodies bound to more than one glycine receptor polypeptide: mAb GlyR 4a stained the 48,000-and 58,000-dalton polypeptides, and mAb GlyR 7a stained the 48,000-and 93,000-dalton polypeptides. Common antigenic determinants thus are shared by the different subunits of the glycine receptor. Complementary results were obtained by peptide mapping of '251-labeled glycine receptor polypeptides with various proteases. A set of peptide fragments of the same apparent molecular mass was produced from the different glycine receptor polypeptides by using V8 protease, chymotrypsin, and elastase. These data suggest that the subunits of the glycine receptor have significant homology within their primary structure and may have evolved from a common ancestor receptor polypeptide.The amino acid glycine is a major inhibitory neurotransmitter in the spinal cord and other regions of the central nervous system of vertebrates (1). Upon binding to its receptor, glycine increases the chloride conductance of the postsynaptic membrane and thus produces a hyperpolarization-i.e., inhibition of neuronal firing (2, 3). This hyperpolarizing action of glycine can be antagonized by the plant alkaloid strychnine, a potent convulsive poison in man and animals (1-4). Radiolabeled strychnine has been widely used to study and localize the glycine receptor in the nervous systems of different species (5, 6). We have employed strychnine as a tool in the purification and biochemical analysis of the glycine receptor (reviewed in ref. 7).From electrophysiological and biochemical studies, it was concluded that the glycine receptor is an integral membrane protein that contains a chemically gated chloride ion channel. Upon solubilization with nonionic detergents, the glycine receptor behaves as a large glycoprotein (molecular mass -250 kDa) whose native conformation depends on the presence of exogenous phospholipids (8, 9). After affinitypurification on aminostrychnine-agarose, three polypeptides of 48, 58, and 93 kDa are detected (9); all three polypeptides are thought to be subunits of the glycine receptor (7, 9). The 48-kDa polypeptide can be covalently labeled with [3H]-strychnine and thus harbors the antagonist binding site of the glycine receptor (10,11). The function of the other polypeptides is presently unknown.To further explore the membrane topology and functional sites of the different glycine receptor subunits, we have produced monoclonal antibodies (mAbs) against this neuronal membrane protein. Here, we report that two of these mAbs recognize mor...

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.