Characterization of distinct single-channel properties of Ca2+ inward currents in mitochondria

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca2+ uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca2+ currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca2+ as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca2+ in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K+-channel inhibitors, and Cl- channel blockers disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca2+ currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca2+ uniporter.