Mitochondrial free [Ca(2+)] dynamics measured with a novel low-Ca(2+) affinity aequorin probe.
Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca(2+)] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca(2+) to permeabilized HeLa cells triggers an increase in [Ca(2+)](M) up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5-1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca(2+)] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca(2+)](M) levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca(2+) uniporter largely reduced the rate of [Ca(2+)](M) increase, but the final steady-state [Ca(2+)](M) reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca(2+)](M) without problems derived from aequorin consumption.
Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, Spain.
Sergio
S
de la Fuente
Rosalba I
RI
Fonteriz
Pedro J
PJ
de la Cruz
Mayte
M
Montero
Javier
J
Alvarez
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D061209:Proton Ionophores / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
D015220:Calcium Channels; D015335:Molecular Probes; D061209:Proton Ionophores; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C034703:mitochondrial calcium uniporter; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000331:Aequorin; D002118:Calcium