title: 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.
abstract: The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) 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 Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), 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 Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.
authors.affiliation: Department of Pharmacology and Toxicology, Karl Franzens University, Graz A-8010, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: M
authors.initials: M
authors.lastname: Poteser
authors.forename: C
authors.initials: C
authors.lastname: Romanin
authors.forename: H
authors.initials: H
authors.lastname: Kahr
authors.forename: M X
authors.initials: MX
authors.lastname: Zhu
authors.forename: K
authors.initials: K
authors.lastname: Groschner
mesh_terms: D000878:Anthranilate Synthase / Q000378:metabolism*; D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D004048:Diethylamines / Q000494:pharmacology; D006801:Humans; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009589:Nitrogen Oxides; D029701:Saccharomyces cerevisiae Proteins; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D004048:Diethylamines; D009097:Multienzyme Complexes; D009589:Nitrogen Oxides; D029701:Saccharomyces cerevisiae Proteins; C032456:antimycin; D009569:Nitric Oxide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000968:Antimycin A; D019284:Thapsigargin; C087229:diethylamine dinitric oxide adduct; D007209:Indole-3-Glycerol-Phosphate Synthase; D000878:Anthranilate Synthase; C087439:TRP3 protein, S cerevisiae; D002118:Calcium