title: Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation.
abstract: In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.
authors.affiliation: Department of Medical Biochemistry & Medical Molecular Biology, University of Graz, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
authors.forename: Maud
authors.initials: M
authors.lastname: Frieden
authors.forename: Karin
authors.initials: K
authors.lastname: Osibow
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
mesh_terms: D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*
chemical_list: D002021:Buffers; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D006632:Histamine; D002118:Calcium