Slow Ca(2+) Efflux by Ca(2+)/H(+) Exchange in Cardiac Mitochondria Is Modulated by Ca(2+) Re-uptake via MCU, Extra-Mitochondrial pH, and H(+) Pumping by F(O)F(1)-ATPase

Mitochondrial (m) Ca(2+) influx is largely dependent on membrane potential (ΔΨ(m)), whereas mCa(2+) efflux occurs primarily via Ca(2+) ion exchangers. We probed the kinetics of Ca(2+)/H(+) exchange (CHE(m)) in guinea pig cardiac muscle mitochondria. We tested if net mCa(2+) flux is altered during a matrix inward H(+) leak that is dependent on matrix H(+) pumping by ATP(m) hydrolysis at complex V (F(O)F(1)-ATPase). We measured [Ca(2+)](m), extra-mitochondrial (e) [Ca(2+)](e), ΔΨ(m), pH(m), pH(e), NADH, respiration, ADP/ATP ratios, and total [ATP](m) in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca(2+) after adding DNP and CaCl(2) is dependent on whether the ΔpH(m) gradient is/is not maintained by reciprocal outward H(+) pumping by complex V. We found that adding CaCl(2) enhanced DNP-induced increases in respiration and decreases in ΔΨ(m) while [ATP](m) decreased, ΔpH(m) gradient was maintained, and [Ca(2+)](m) continued to increase slowly, indicating net mCa(2+) influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl(2) caused larger declines in ΔΨ(m) as well as a slow fall in pH(m) to near pH(e) while [Ca(2+)](m) continued to decrease slowly, indicating net mCa(2+) efflux in exchange for H(+) influx (CHE(m)) until the ΔpH(m) gradient was abolished. The kinetics of slow mCa(2+) efflux with slow H(+) influx via CHE(m) was also observed at pH(e) 6.9 vs. 7.6 by the slow fall in pH(m) until ΔpH(m) was abolished; if Ca(2+) reuptake via the MCU was also blocked, mCa(2+) efflux via CHE(m) became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHE(m) activity is driven largely by the ΔpH(m) chemical gradient with H(+) leak, while mCa(2+) entry via MCU depends largely on the charge gradient ΔΨ(m). A fall in ΔΨ(m) with excess mCa(2+) loading can occur during cardiac cell stress. Cardiac cell injury due to mCa(2+) overload may be reduced by temporarily inhibiting F(O)F(1)-ATPase from pumping H(+) due to ΔΨ(m) depolarization. This action would prevent additional slow mCa(2+) loading via MCU and permit activation of CHE(m) to mediate efflux of mCa(2+). HIGHLIGHTS: -. We examined how slow mitochondrial (m) Ca(2+) efflux via Ca(2+)/H(+) exchange (CHE(m)) is triggered by matrix acidity after a rapid increase in [Ca(2+)](m) by adding CaCl(2) in the presence of dinitrophenol (DNP) to permit H(+) influx, and oligomycin (OMN) to block H(+) pumping via F(O)F(1)-ATP synthase/ase (complex V). -. Declines in ΔΨ(m) and pH(m) after DNP and added CaCl(2) were larger when complex V was blocked. -. [Ca(2+)](m) slowly increased despite a fall in ΔΨ(m) but maintained pH(m) when H(+) pumping by complex V was permitted. -. [Ca(2+)](m) slowly decreased and external [Ca(2+)](e) increased with declines in both ΔΨ(m) and pH(m) when complex V was blocked. -. ATP(m) hydrolysis supports a falling pH(m) and redox state and promotes a slow increase in [Ca(2+)](m). -. After rapid Ca(2+) influx due to a bolus of CaCl(2), slow mCa(2+) efflux by CHE(m) occurs directly if pH(e) is low.