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

Helen M. Brereton, M. Lyn Harland, Amanda M. Auld, Greg J. Barritt

    Research output: Contribution to journalArticle

    34 Citations (Scopus)

    Abstract

    The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca2+ channel, in storeoperated 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-1 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 μM), 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+ inflow 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 sacs 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.

    Original languageEnglish
    Pages (from-to)63-74
    Number of pages12
    JournalMOLECULAR AND CELLULAR BIOCHEMISTRY
    Volume214
    Issue number1
    DOIs
    Publication statusPublished - Nov 2000

    Keywords

    • Antisense
    • Non-selective cation channels
    • Polyclonal antibody
    • TRP channels
    • Xenopus laevis oocytes

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