Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation

Abstract

In endothelial cells, local Ca2+ release from superficial endoplasmic reticulum (ER) activates BKca channels. The resulting hyperpolarization promotes capacitative Ca2+ entry (CCE), which, unlike BKCa channels, is inhibited by high Ca2+. To understand how the coordinated activation of plasma membrane ion channels with opposite Ca2+ sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca2+ concentration ([Ca2+]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 BKCa channels was used to estimate local [Ca2+]pm. Under standard patch conditions (130 mM K+ in the bath), histamine increased [Ca2+]pm at L1 and L3 to ∼1.6 μM, whereas close to mitochondria [Ca2+]pm remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca2+]pm at L2 increased in response to histamine. Under standard patch conditions Ca2+ entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mM K+ in the bath), changes in [Ca2+]pm to histamine could be monitored without cell depolarization and, thus, in conditions where Ca2+ entry occurred. Here, histamine induced an initial transient Ca2+ elevation to ≥3.5 μM followed by a long lasting plateau at ∼1.2 μM in L1 and L3, whereas mitochondria kept neighboring [Ca2+]pm low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca2+ allowing simultaneous activation of BKCa and CCE, despite their opposite Ca2+ sensitivity.