Cloning of Trp1 beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

The Trp gene product has been proposed as a candidate protein for the store -operated Ca2+ channel, but the Trp protein(s) has not been identified in a ny nonexcitable cell. We report here the cloning of a rat brain Trp1 beta c DNA and detection and immunolocalization of the endogenous and expressed Tr p1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplifi ed from rat submandibular gland RNA. Rat-specific primers were used for clo ning of a full-length rat brain Trp1 beta cDNA(rTrp1), encoding a protein o f 759 amino acids. Northern blot analysis demonstrated the transcript in se veral rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunopr ecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted wi th a protein of similar to 92 kDa, but not with hTrp3, in membranes of hTrp 3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa i n human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 prote ins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first tim e the presence of Trp1 protein in a nonexcitable cell.