Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

• Published in: Molecular and cellular biochemistry 2000-11, Vol.214 (1-2), p.63-74
• Main Authors: Brereton, H M, Harland, M L, Auld, A M, Barritt, G J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Base Sequence; Calcium - metabolism; Calcium Channels - metabolism; DNA Primers - genetics; DNA, Complementary - genetics; Drosophila; Female; Gene Expression; Humans; In Vitro Techniques; Inflow; Lanthanides; Molecular Sequence Data; Oocytes - metabolism; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Antisense - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; TRPC Cation Channels; Water inflow; Xenopus laevis
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1023/A:1007193720930

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in store-operated Ca2+ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3' and 5' rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-l polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca2+ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP3F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC50 = 0.5 microM), indicating that InsP3F and LPA principally activate store-operated Ca2+ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP3F-, LPA- or thapsigargin-stimulated Ca2+ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP3F-stimulated Ca2+ inflow and only a very small enhancement of LPA-stimulated Ca2+ in-flow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca2+ inflow through the previously-characterised Ca2+ -specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.