Reprogramming of Mitochondrial Ca2+ Handling in MICU1-Deficient HeLa Cells

Abstract

Recent studies have revealed MCU as the pore forming domain and MICU1 as a critical Ca2+-sensitive regulator of the mitochondrial Ca2+ uniporter. However, the exact role of MICU1 in Ca2+ transport remains to be addressed. Our previous studies showed that prolonged down-regulation of MICU1 in HeLa cells (shMICU1) promotes mitochondrial Ca2+ uptake at low [Ca2+], which unexpectedly, fails to effectively increase matrix [Ca2+]. To determine the source of discrepancy between the mitochondrial Ca2+ uptake and the matrix [Ca2+] phenotypes, first we simultaneously monitored ruthenium red-sensitive clearance of added Ca2+ from the cytoplasm and the corresponding matrix [Ca2+] response in permeabilized shMICU1 cells. Under conditions of similar cytoplasmic Ca2+ clearance, shMICU1 cells showed a smaller matrix [Ca2+] increase than the control, indicating enhanced buffering of Ca2+ in the matrix. Enhanced Ca2+ binding in the matrix likely reflects alkalinization and enhanced phosphate transport. To test if upregulation of Ca2+ buffering is directly linked to MICU1 depletion, we also assessed mitochondrial Ca2+ handling after 72hr silencing of MICU1 (siMICU1). In siMICU1cells both mitochondrial Ca2+ uptake and the matrix [Ca2+] rise were effectively stimulated at low Ca2+ levels. Thus, upregulation of matrix Ca2+ buffering seems to be a component of an adaptive response to sensitization of mitochondrial Ca2+ uptake in shMICU1. The adaptive response is likely to be important to attenuate some MICU1-depletion induced cellular impairments that we found to manifest as attenuated mitochondrial ATP production and cell proliferation.