CELLULAR AND SUBCELLAR CALCIUM ION HOMEOSTASIS

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This project focuses on the mechanism whereby cells achieve homeostasis of calcium ion concentration, both within the cytosol and other cellular compartments, and allow changes in calcium in response to hormones and neurotransmitters. This year we have taken advantage of the availability of membrane-permanent fluorescent calcium ion-chelating agents to measure intramitochondrial free calcium concentrations both in suspensions of heart mitochondria and, in a collaboration with the Cardiac Function Section, in situ in a single cardiac myocyte. In the work with isolated mitochondrial membrane we have measured the concentration gradient of calcium ions across the mitochondrial membrane, with respect to varying extramitochondrial calcium and also with respect to changes in the transmembrane pH gradient, which provides the driving force for calcium afflux from the mitochondria. Under simulated physiological conditions, i.e., in the presence of cytosolic concentrations of sodium and magnesium ions, respiring mitochondria were shown to maintain a negative calcium gradient (inside divided by outside) when extramitochondrial calcium was less than 500 nM: at higher values than this, the mitochondrial calcium concentration gradient becomes positive. The collaborative studies on intramitochondrial calcium in situ in a single living cell are quite novel and have shown that this parameter lies within the range previously shown to modulate intramitochondrial dehydrogenase activity. Further, intramitochondiral free calcium rises in response to beta-adrenergic stimulation and increased rate of electrical stimulation, situations which enhance work output by the cell and thus require more active oxidative phosphorylation by the mitochondria.