The postsynaptic glycine receptor of rat spinal cord is a glycosylated membrane protein that, after affinity purification, contains membrane-spanning subunits of Mr 48,000 and 58,000 and an associated peripheral polypeptide of Mr 93,000. Here, the quaternary structure of the transmembrane core of the receptor was investigated by chemically crosslinking its subunits. Upon treatment with crosslinking reagents of different side-chain specificities and lengths, a consistent set of adducts up to M, 260,000 was detected after separation by NaDodSO4/PAGE. The observed pattern of adducts was similar irrespective of whether purified receptor protein or synaptosomal membranes were crosslinked. Compositional Recent results obtained by cDNA cloning of subunits of the nicotinic acetylcholine (1-3), the glycine (4), and the yaminobutyric acid (5) receptors revealed significant homology in primary structure and predicted transmembrane topology between these chemically gated ion channel proteins. In particular, four hydrophobic segments (M1-M4) presumed to span the lipid bilayer are conserved in the subunits of the different receptors. These data established the existence of a previously postulated superfamily ofphylogenetically related ion channel-forming receptor polypeptides (6, 7).Electron microscopy (8, 9) and crosslinking experiments (10) have demonstrated that the four subunits of Torpedo and muscle nicotinic acetylcholine receptor (nAcChoR) are arranged in a quasisymmetrical pentameric (a2f3y5) structure around a central pit presumed to represent the transmembrane channel. Little, however, is known about stoichiometry, spatial arrangement, and total number of subunits constituting neuronal ligand-gated ion channels. The postsynaptic y-aminobutyric acid receptor (GABAAR) has been proposed to be a tetrameric transmembrane protein (11). For neuronal nAcChoR, tetrameric and pentameric structures are discussed (12, 13).Another member of the chemically gated ion channel family, the glycine receptor (GlyR), mediates postsynaptic inhibition in spinal cord and other parts ofthe central nervous system (14). Binding of the agonist glycine induces an increase in chloride permeability of the postsynaptic membrane and thus antagonizes depolarization-i.e., reduces neuronal firing rate (15). The convulsive alkaloid strychnine antagonizesglycine-mediatedinhibition,andglycine-displaceable [3H]strychnine binding has been used to investigate and localize the GlyR in the nervous system of different species (16)(17)(18)(19). The GlyR has been purified from spinal cord of different mammals by affinity chromatography on aminostrychnine-agarose and shown to be a large glycoprotein containing polypeptides of Mr 48,000 (a), 58,000 ((3), and 93,000 (y) (20)(21)(22). The Mr 48,000 subunit can be covalently labeled with [3Hlstrychnine by UV illumination and thus contains the antagonist-binding site of the GlyR (23). The Mr 48,000 and 58,000 polypeptides are glycosylated (21, 24) and assumed to be homologous on the basis of immunological cr...

The quaternary structure and functional properties of synaptophysin, a major integral membrane protein of small presynaptic vesicles, were investigated. Cross-linking and sedimentation studies indicate that synaptophysin is a hexameric homo-oligomer, which in electron micrographs exhibits structural features common to channel-forming proteins. On reconstitution into planar lipid bilayers, purified synaptophysin displays voltage-sensitive channel activity with an average conductance of about 150 picosiemens. Because specific channels and fusion pores have been implicated in vesicular uptake and release of secretory compounds, synaptophysin may have a role in these processes.

Hybridomas producing monoclonal antibodies to Rickettsia rickettsii were prepared from mice to investigate the function of rickettsial antigens. Of the 31 reactive hybridoma lines thus far tested for immunoglobulin subclasses, 11 belonged to the IgG2A subclass, 9 to the IgG2B subclass, and 7 to the IgG3 subclass; four did not react with any of the isotyping sera. Five of the antibodies recognized epitopes present on molecules that were presumed to be polysaccharide and heterogeneous in molecular weight. Twenty monoclonal antibodies reacted with a 170,000-dalton antigen, and six precipitated both the 133,000- and 32,000-dalton polypeptides. Only those antibodies to the 170,000- and 133,000-dalton antigens protected mice from challenge with R. rickettsii. Antibodies to these same antigens were detected in sera from patients convalescing from Rocky Mountain spotted fever. All monoclonal antibodies reacted with antigens apparently located on the rickettsial surface. The protective activity of these antibodies was not correlated with their reactivity in complement fixation, enzyme-linked immunosorbent assay, and immunofluorescence tests.

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