Control of Mitochondrial Ca2+ Uptake Threshold Via the Micu1:Mcu Ratio

Mitochondrial Ca2+ uptake mediated by the Ca2+ uniporter plays important effector and modulator roles in cellular Ca2+ signaling. The activation of the uniporter generally requires cytosolic [Ca2+] ([Ca2+]c) >1μM; however, in certain cell types i is activated at much lower [Ca2+]c. We have recently identified the EF-hand protein MICU1 being responsible for both the relatively high threshold and positive cooperativity of the uniporter's activation by [Ca2+]c. Here we examined the idea that the cell-type-dependent differences in the [Ca2+]c threshold for uniporter activation reflected differences in the relative availability of MICU1 to the pore protein MCU. First, we tested if decreasing the MICU1:MCU ratio via transient overexpression of MCU could lower the threshold for Ca2+ uptake in HeLa cells. Simultaneous fluorescence imaging of [Ca2+]c and [Ca2+] in the mitochondrial matrix ([Ca2+]m) confirmed a shorter coupling time between store-operated entry-derived [Ca2+]c and [Ca2+]m rises for the cells overexpressing MCU. Furthermore, upon permeabilization, elevated [Ca2+]m baseline and robust enhancement of [Ca2+]m response to small [Ca2+]c rises was observed in MCU overexpressing cells. Importantly, overexpression of both MCU and MICU1 did not result sensitization of mitochondria to low [Ca2+]c levels. Next, we compared MICU1 and MCU expression levels in H295R human adrenal carcinoma cells (threshold ∼250nM) vs. Hela cells (threshold ∼1μM) and in rat INS1 insulinoma cells (threshold ∼190nM) vs. RBL2H3 mast cells (threshold ∼1μM). Despite displaying a MICU1-deficient like [Ca2+]m phenotype, H295R and INS1 cells their MICU1 expression was relatively strong and even more, their MICU1:MCU mRNA ratio was high. Thus, shifting the ratio of MICU1:MCU is an effective way of [Ca2+]c threshold tuning for the uniporter in HeLa cells. However, threshold variances between cell types do not necessarily reflect MICU1:MCU differences but rather other alterations in the uniporter channel complex.