EPITHELIAL INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS - MULTIPLICITY OF LOCALIZATION, SOLUBILITY, AND ISOFORMS

In an earlier subcellular fractionation study of epithelial tissue (li ver and pancreas), we demonstrated that the inositol 1,4,5-trisphospha te receptor (IP(3)R) is found in association with biochemically distin ct cellular membranes, including the endoplasmic reticulum (ER) and pl asma membrane (Sharp, A H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP(3)R s, we have now employed cultured Madin-Darby canine kidney (MDCK) cell s, a well studied tight polarized epithelial cell type. Indirect immun ofluorescence with an antiserum which specifically recognizes IP(3)R i n MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane like staini ng pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral protei ns (Na+,K+-ATPase and the facilitated glucose trans porter) gave a con tinuous basolateral staining pattern, that seen for IP(3)R was discont inuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolater al plasma membrane-like staining pattern observed for IP(3)R reflects its localization to basolateral caveolae. Biotinylation of non-permeab ilized and permeabilized MDCK cells, followed by immunoprecipitation o f IP(3)R and detection with streptavidin, indicated that while most IP (3)R is localized to biotin-inaccessible compartments (i.e. ER), a fra ction (10-20%) of IP(3)R was accessible to externally added biotin pri marily from the basolateral side. This result is compatible with the d ual ER and basolateral caveolar localization suggested by immunocytoch emistry, although it does not exclude the presence of some IP(3)R in t he basolateral plasma membrane as well. Solubility studies revealed IP (3)R to be considerably more insoluble than the basolateral proteins, Na+,K+-ATPase and the liver cell adhesion molecule, as well as the cyt oskeletal proteins, ankyrin and fodrin. In the most insoluble fraction , IP(3)R was found along with caveolin, further supporting the notion that part of the cellular IP(3)R pool associates with caveolae. Since multiple localizations of IP(3)R within a cell might reflect the exist ence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP(3 )R was performed. All three isoforms of IP(3)R were expressed in the h omogeneous population of MDCK cells. The existence of distinct membran e localizations and multiple isoforms of IP(3)R within the same cell t ype suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools i n epithelial and other cells.