MICU1 Serves as a Ca2+-Controlled Gatekeeper for the Mitochondrial Ca2+ Uniporter

Abstract

MICU1 serves as a Ca2+-controlled gatekeeper for the mitochondrial Ca2+ uniporter Mitochondria are important targets and relay points of calcium signaling. Ca2+ is driven to the mitochondrial matrix via a low-affinity Ca2+ channel, the Ca2+ uniporter (MCU) that exhibits time-dependent sensitization by Ca2+. However, the molecular mechanism of the MCU's regulation by Ca2+ remains unclear. Recently, MICU1 has been identified as an EF-hand-containing regulatory component of the MCU. We set out to elucidate the role of MICU1 in the Ca2+ regulation of the MCU and to examine its possible contribution to the sensitization. Mitochondrial Ca2+ uptake was evaluated fluorometrically in suspensions of permeabilized MICU1 knockdown (MICU1KD) and control HeLa cells as ruthenium red-sensitive Ca2+ clearance from the incubation buffer. Clearance of small Ca2+ pulses elevating the [Ca2+] to 10uM were taken up effectively by both MICU1KD and control cells. The dose-response for the clearance of added Ca2+ was sigmoidal in the control cells. This was leftward-shifted and showed lesser cooperativity in the MICU1KD cells. MICU1KD rescue with wild type MICU1 restored the control type dose-response, whereas an EF-hand-mutant MICU1 shifted it to the right. When the time-dependent sensitization of the MCU was tested by a two-pulse protocol, the MICU1KD failed to show a further increase in Ca2+ sensitivity. As to the functional significance of altered Ca2+ handling in MICU1KD, these cells displayed lesser Ca2+ tolerance and more cell death under stress conditions. We propose that the MICU1, through its EF-hands, controls the [Ca2+] set-point for MCU channel closure. MICU1 helps mitochondria to respond to physiological [Ca2+] oscillations and provides some protection from Ca2+ overloading.