Involvement of trp-2 protein in store-operated influx of calcium in fibroblasts

Mammalian homologues of the Drosophila melanogaster transient receptor pote ntial (trp) gene have been proposed to encode store-operated channels. This assertion essentially stays on the fact that expression of different trp p roteins produces trans-membrane cation currents. However, the selectivity o f the expressed channels and their mode of activation, in particular, their dependence to store depletion appears to be quite variable. In the present work, we adopted an anti-sense strategy to study this question in transfec ted Chinese hamster ovary cells expressing the rat neurotensin receptor (CH O-NTR cells), a cellular model characterized by its very large store-depend ent entry of Ca2+. We identified different trp transcripts by RT-PCR, the t rp-1 and trp-2 transcripts being by far the most abundant. CHO-NTR cells we re then transfected with a mouse trp-2 anti-sense construct (CHO-NTR-TRP2AS cells). We showed that in these cells, trp-2 mRNA was suppressed in compar ison with cells transfected with a control plasmid. The store-operated entr y of Ca2+ was evaluated after store depletion by an IP3-dependent mechanism (neurotensin stimulation) or by direct inhibition of the endoplasmic retic ulum Ca(2+)ATPase (thapsigargin stimulation). In both cases, store-dependen t entry of Ca2+ was largely reduced in CHO-NTR-TRP2AS cells in comparison w ith control cells, suggesting that trp-2 protein might constitute a functio nal subunit of store-operated channels. (C) 2001 Harcourt Publishers Ltd.