MICU2基因敲除HEK293细胞

OBJECTIVE:Transport of Ca into pancreatic β cell mitochondria facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca uniporter (MCU) and associated proteins allows modification of mitochondrial Ca transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets. METHODS:siRNA silencing of Micu2 in the INS-1 832/13 and EndoC-βH1 cell lines was performed; Micu2 mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca dynamics were performed. RESULTS:Silencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-βH1 cells. The Micu2 mice also displayed attenuated GSIS. Mitochondrial Ca uptake declined in MICU2-deficient INS-1 832/13 and EndoC-βH1 cells in response to high glucose and high K. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca uptake, cytosolic Ca was lower in siMICU2-treated INS-1 832/13 cells in response to high K. It was hypothesized that Ca accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca channels, lowering total cytosolic Ca. Upon high K stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca levels. CONCLUSIONS:MICU2 plays a critical role in β cell mitochondrial Ca uptake. β cell mitochondria sequestered Ca from the submembrane compartment, preventing desensitization of voltage-dependent Ca channels and facilitating GSIS.