The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells

We utilized 2-aminoethyoxydiphenyl borane, an agent that blocks store-operated Ca2+ entry, as well as an antisense approach to characterize endogenous Ca2+ entry pathways in HEK-293 cells. The thapsigargin- and carbachol-induced, but not the 1-oleolyl-2-acytyl-sn-glycerol (OAG).induced, entry was blocked by 2-aminoethyoxydiphenyl borane. Both reverse transcriptase-PCR and Western blot analyses demonstrated endogenous expression for HTRP1, HTRP3, and HTRP4 and specific suppression of mRNA levels and Trp protein levels in cells stably expressing antisense constructs. Expression of HTRP4 antisense inhibited 35% of the carbachol (CCh)-stimulated Ba2+ entry and 46% of the OAG-stimulated Sr2+ entry but in contrast had no effect on the thapsigargin-stimulated Ba2+ or Sr2+ entry. HTRP3 antisense reduced, while HTRP1 antisense had no effect on, OAG-induced Sr2+ entry. Of greater importance, HTRP4 antisense expression, but not HTRP3 antisense expression, blocked the sustained Ca2+ oscillations produced by low doses of CCh (15 muM), arguing that receptor-stimulated rather than store-operated channels are involved in these sustained oscillations. HTRP4 antisense also inhibited 75% of the arachidonic acid-induced Ca2+ entry. In summary, these data suggest that HTRP4 proteins in HEK-293 cells, differing from HTRP3 and HTRP1 proteins, do not serve as functional subunits of store-operated channels but do function as subunits for CCh- and OAG-stimulated channels. Furthermore, evidence is provided for the first time for the involvement of a Trp isoform (HTRP4) in the formation of the channel responsible for both arachidonic acid-induced Ca2+ entry and the Ca2+ entry needed to sustain long term Ca2+ oscillations induced by low doses of carbachol.