In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining 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 proteins (Na+,K+-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K+-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology