Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling -: Evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels

The role of Trp3 in cellular regulation of Ca2+ entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca2+ entry (CCE)-mediated) intracellular Ca2+ signals and Mn2+ entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 muM) or by authentic NO (100 muM). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca2+ signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn2+ entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn2+ entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca2+, which was not detectable in controls. Suppression of mitochondrial Ca2+ handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nM) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca2+ entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca2+ entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca2+ and membrane currents revealed that Trp3 cation currents are inhibited during Ca2+ entry-induced elevation of cytoplasmic Ca2+, and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca2+ handling. Thus, we propose Trp3 as a subunit of CCE channels.