TRANSMEMBRANE TOPOLOGY AND SITES OF N-GLYCOSYLATION OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR

To define the transmembrane topology of the inositol 1,4,5-trisphospha te receptor (InsP(3)R), we determined the subcellular location of the hydrophilic segment (residues 2463-2529 of mouse type 1 InsP(3)R) beli eved to be located at the luminal side of the endoplasmic reticulum (E R) in the six-transmembrane model but at the cytoplasmic side in the e ight-transmembrane model. This hydrophilic segment includes two consen sus sites for N-glycosylation (Asn-2475 and Asn-2503). We prepared an anti-peptide antibody against residues 2504-2523. Electron microscope immunocytochemical studies of mouse cerebellar Purkinje cells showed t hat binding of this antibody frequently occurs in the intracisternal s pace of the ER. We constructed three mutant receptors by site-directed mutagenesis of Asn to Gin (N2475Q, N2503Q, and N2475and/N2503Q), By c oncanavalin A column chromatography of these receptors, we found that both Asn-2475 and Asn-2503 are glycosylated. These results indicate th at residues 2504-2523, Asn-2475, and Asn-2503 are exposed to the ER lu men. We therefore propose that InsP(3)R has six membrane spanning segm ents. Based on the transmembrane topology and subunit organization, we suggest that InsP(3)R is a member of the superfamily that includes th e voltage- and second messenger-gated ion channels on the plasma membr ane.