Spontaneous Ca²⁺ signaling from the InsP₃R intracellular Ca²⁺ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca²⁺ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP₃R activity or mitochondrial Ca²⁺ uptake increased α-ketoglutarate (αKG) abundance and the NAD⁺/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)–to–mitochondria Ca²⁺ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca²⁺ inhibited αKGDH activity and increased NAD⁺, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca²⁺ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca²⁺ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca²⁺ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

从 InsP ₃ R 细胞内 Ca ²⁺释放通道到线粒体的自发 Ca ²⁺信号传导对于最佳氧化磷酸化 (OXPHOS) 和 ATP 产生至关重要。在 OXPHOS 缺陷的细胞中，还原羧化取代了氧化代谢，以维持还原当量和代谢前体的数量。为了研究线粒体 Ca ²⁺摄取在调节这些细胞中的生物能量学中的作用，我们使用了 OXPHOS 感受态和 OXPHOS 缺陷细胞。抑制 InsP ₃ R 活性或线粒体 Ca ²⁺摄取增加 α-酮戊二酸 (αKG) 丰度和 NAD ⁺/NADH 比率，表明组成型内质网 (ER) 到线粒体 Ca ²⁺转移促进了最佳的 αKG 脱氢酶 (αKGDH) 活性。减少线粒体 Ca ²⁺可抑制 αKGDH 活性并增加 NAD ⁺，从而在 OXPHOS 感受态细胞和 OXPHOS 缺陷细胞中诱导 SIRT1 依赖性自噬。虽然 OXPHOS 感受态细胞中的自噬通量促进细胞存活，但由于自噬体-溶酶体融合的抑制，它在 OXPHOS 缺陷细胞中受损。抑制 αKGDH 和 OXPHOS 缺陷细胞中的自噬通量受损导致细胞死亡，以响应 Ca ^{2+组成通量的中断}从 ER 到线粒体。这些结果表明，线粒体在维持 OXPHOS 感受态细胞和 OXPHOS 缺陷细胞的生物能量稳态中发挥着重要作用，其中 Ca ²⁺对 αKGDH 活性的调节起着关键作用。抑制 ER 到线粒体的 Ca ²⁺转移可能代表一种针对癌细胞的一般治疗策略，无论其 OXPHOS 状态如何。