Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

1 Secretory Physiology Section, 2 Gene Regulation and Expression Unit, Gene Therapy and Therapeutics Branch, and 3 Imaging Core Facility, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892; 4 Department of Anesthesiology and 6 Departments of Anesthesiology, Biological Chemistry, and Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90024; and 5 Department of Pharmacology and Neurobiotechnology Center, Ohio State University, Columbus, Ohio, 43210 The Trp gene product has been proposed as a candidate protein for the store-operated Ca 2+ channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1 cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1 cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1 ) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2 ) reacted with a protein of ~92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3 ) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca 2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell. store-operated calcium channel; Trp protein; plasma membrane; nonexcitable cells