Data_Sheet_1_Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase.docx

Mitochondrial (m) Ca2+ influx is largely dependent on membrane potential (ΔΨm), whereas mCa2+ efflux occurs primarily via Ca2+ ion exchangers. We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa2+ flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATPm hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+]m, extra-mitochondrial (e) [Ca2+]e, ΔΨm, pHm, pHe, 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 Ca2+ after adding DNP and CaCl2 is dependent on whether the ΔpHm gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in ΔΨm while [ATP]m decreased, ΔpHm gradient was maintained, and [Ca2+]m continued to increase slowly, indicating net mCa2+ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in ΔΨm as well as a slow fall in pHm to near pHe while [Ca2+]m continued to decrease slowly, indicating net mCa2+ efflux in exchange for H+ influx (CHEm) until the ΔpHm gradient was abolished. The kinetics of slow mCa2+ efflux with slow H+ influx via CHEm was also observed at pHe 6.9 vs. 7.6 by the slow fall in pHm until ΔpHm was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa2+ efflux via CHEm became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the ΔpHm chemical gradient with H+ leak, while mCa2+ entry via MCU depends largely on the charge gradient ΔΨm. A fall in ΔΨm with excess mCa2+ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa2+ overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to ΔΨm depolarization. This action would prevent additional slow mCa2+ loading via MCU and permit activation of CHEm to mediate efflux of mCa2+.HIGHLIGHTS-We examined how slow mitochondrial (m) Ca2+ efflux via Ca2+/H+ exchange (CHEm) is triggered by matrix acidity after a rapid increase in [Ca2+]m by adding CaCl2 in the presence of dinitrophenol (DNP) to permit H+ influx, and oligomycin (OMN) to block H+ pumping via FOF1-ATP synthase/ase (complex V).-Declines in ΔΨm and pHm after DNP and added CaCl2 were larger when complex V was blocked.-[Ca2+]m slowly increased despite a fall in ΔΨm but maintained pHm when H+ pumping by complex V was permitted.-[Ca2+]m slowly decreased and external [Ca2+]e increased with declines in both ΔΨm and pHm when complex V was blocked.-ATPm hydrolysis supports a falling pHm and redox state and promotes a slow increase in [Ca2+]m.-After rapid Ca2+ influx due to a bolus of CaCl2, slow mCa2+ efflux by CHEm occurs directly if pHe is low.

线粒体（m）Ca2+的内流主要依赖于膜电位（ΔΨm），而mCa2+的外流则主要通过Ca2+离子交换器进行。本研究探讨了在豚鼠心肌线粒体中，Ca2+/H+交换（CHEm）的动力学。我们检验了在依赖于线粒体基质H+泵送（由ATPm水解在复合物V（FOF1-ATPase）驱动）的基质内向H+泄漏期间，净mCa2+通量是否发生改变。我们测量了[Ca2+]m、细胞外（e）[Ca2+]e、ΔΨm、pHm、pHe、NADH、呼吸、ADP/ATP比率以及存在或不存在质子载体二硝基酚（DNP）、线粒体单一转运蛋白（MCU）阻断剂Ru360和复合物V阻断剂寡霉素（OMN）时的总[ATP]m。我们提出，在添加DNP和CaCl2后，净慢速的Ca2+内流/外流取决于ΔpHm梯度是否通过复合物V的相互逆向H+泵送得以维持。我们发现，添加CaCl2增强了DNP诱导的呼吸增加和ΔΨm下降，同时[ATP]m减少，ΔpHm梯度得以维持，[Ca2+]m持续缓慢增加，表明通过MCU的净mCa2+内流。相反，当复合物V被OMN阻断时，添加DNP和CaCl2导致ΔΨm更大程度地下降，以及pHm缓慢下降至接近pHe，而[Ca2+]m持续缓慢下降，表明通过交换H+流入（CHEm）的净mCa2+外流，直到ΔpHm梯度被消除。在pHe 6.9与7.6处，通过pHm的缓慢下降直至ΔpHm被消除，也观察到了通过CHEm的慢速H+流入的慢速mCa2+外流动力学；如果通过MCU的Ca2+重摄取也被阻断，mCa2+通过CHEm的外流变得更加明显。对于质子电化学梯度的两个组成部分，我们的结果表明，CHEm活性主要由H+泄漏驱动的ΔpHm化学梯度驱动，而通过MCU的mCa2+进入则主要依赖于电荷梯度ΔΨm。在心脏细胞应激期间，可能发生ΔΨm去极化导致的FOF1-ATPase泵送H+的过度mCa2+加载。通过暂时抑制FOF1-ATPase泵送H+以降低ΔΨm去极化，可以减少由于mCa2+超负荷引起的心脏细胞损伤。这一行动将防止通过MCU的额外慢速mCa2+加载，并允许激活CHEm以介导mCa2+的外流。重点——我们研究了在添加CaCl2以通过DNP允许H+流入并使用寡霉素（OMN）阻断通过FOF1-ATP合酶/酶（复合物V）的H+泵送后，快速增加[Ca2+]m后线粒体（m）Ca2+通过CHEm的慢速外流是如何由基质酸度触发的。——当复合物V被阻断时，DNP和添加的CaCl2导致的ΔΨm和pHm的下降更大。——尽管ΔΨm下降，但[Ca2+]m缓慢增加，当允许复合物V泵送H+时，pHm得以维持。——当复合物V被阻断时，[Ca2+]m缓慢下降，细胞外[Ca2+]e增加，ΔΨm和pHm均下降。——ATPm水解支持pHm和氧化还原状态的下降，并促进[Ca2+]m的缓慢增加。——在由于CaCl2的冲击而引起的快速Ca2+内流之后，如果pHe较低，则通过CHEm的慢速mCa2+外流直接发生。