The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation

Ryan E Yoast,Scott M Emrich,Xuexin Zhang,Ping Xin,Vikas Arige,Trayambak Pathak,J Cory Benson,Martin T Johnson,Ahmed Emam Abdelnaby,Natalia Lakomski,Nadine Hempel,Jung Min Han,Geneviève Dupont,David I Yule,James Sneyd,Mohamed Trebak

doi:10.1016/j.jbc.2021.101174

Abstract

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C–coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release–activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol–mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.

Highlights

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C–coupled receptors to that of cellular bioenergetics
The addition of a bolus 10 μM Ca2+ to the bath of permeabilized parental cells resulted in rapid mitochondrial uptake, which did not occur in mitochondrial Ca2+ uniporter (MCU)-KO cells, 2 J
When we simulate the effects of MCU knockout (MCU-KO), mitochondrial Na+/Ca2+ exchanger (NCLX)-KO, and MCU-KO + NCLX-KO on thapsigargin-activated cytosolic Ca2+ rise, we found that MCU-KO and MCU-KO + NCLX-KO cause similar increases in cytosolic [Ca2+], whereas NCLX-KO leads to a slight decrease (Fig. 11, A–E), in agreement with our experimental data with MCU and previous work with NCLX [48]

Summary

Introduction

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C–coupled receptors to that of cellular bioenergetics. Despite the critical function of MCU-mediated mitochondrial Ca2+ uptake in inhibiting CRAC channel slow CDI and limiting Ca2+ extrusion, MCU deletion leads to a paradoxical enhancement of cytosolic Ca2+ upon receptor stimulation.

Results

Conclusion