Regulation of ATP production: dependence on calcium concentration and respiratory state

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca²⁺] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H₂O₂ production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca²⁺], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca²⁺] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca²⁺] was positively and continuously associated with H₂O₂ production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.