1.A.77 The Mg2+/Ca2+ Uniporter (MCU) Family

Mitochondrial Ca²⁺ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival. Mitochondrial Ca²⁺ uptake occurs via the ruthenium red sensitive Ca²⁺ uniporter (MCU), and efflux occurs via a Na⁺/Ca²⁺ exchanger (mNCX). De Stefani et al. (2011) and Baughman et al. (2011) simultaneously reported the identity of a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a   uniporter regulator that is present in organisms in which mitochondrial Ca²⁺ uptake was demonstrated and whose sequence includes two transmembrane domains.  Short interfering RNA (siRNA) silencing of MCU reduced mitochondrial Ca²⁺ uptake, and MCU overproduction increased the matrix Ca²⁺ concentration evoked by inositol 1,4,5-trisphosphate-generating agonists. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivities of the uniporter 90. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca²⁺ concentration transients when overexpressed. Thus, MCU is the channel responsible for ruthenium-red-sensitive mitochondrial Ca²⁺ uptake. Distant prokaryotic homologues have been identified (A. Lee and MH Saier, unpublished results).  A small (10 KDa) protein with 1 TMS, EMRE, is required for interaction of MCU with MICU1 and 2 (see figure4F in Sancak et al. 2013).  The entire complex includes MICU1, MICU2, EMRE and MCU, and has a molecular size of 480,000 Daltons.  The MICU family has TC# 8.A.44 and functions in regulation.  The EMRE family has TC# 8.A.45 and funtions to interconnect MUC and MICU in the complex (Sancak et al. 2013).

Mitochondrial calcium uniporter, MCU, forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1 (1.A.76.1.1), and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca²⁺ uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MICU1 (TC# 8.A.44.1.1) is an essential component of the MCU system, and serves as the gatekeeper of MCU-mediated Ca²⁺ uptake that is essnetial to prevent Ca²⁺ overload and associated stress (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b). The oligomeric channel can incorporate an inhibitory subunit, MCUb, that exerts a dominant-negative effect on channel formation (Raffaello et al., 2013). Also essential is the EMRE subunit (TC# 8.A.45.1.1) which binds MICU1 via transmembrane helices to control Ca²⁺ transport activity (Tsai et al. 2016). MCUs interact with Miro1 (Q8IXI2), a mitochondrial Rho GTPase that seems to regulate MCU activities (Niescier et al. 2018). Carboxylate-Capped Analogues of Ru265 are prodrugs that inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders (Bigham et al. 2022)

MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retained function but confered resistance to Ru360, the most potent inhibitor of the uniporter (Baughman et al., 2011). MCU homologues and their cytoplasmic regulatory protein partners with two EF-hand motifs, MICU, are both present in many but not all eukaryotes having MCU (Bick et al., 2012). The phylogenies and domain orders of MCU Ca²⁺ channel homologues have been reported (Bick et al., 2012). MCU is involved in the apoptosis in PC12 cells induced by 1-methyl-4-phenylpyridinium ions (MPP) (Wang et al. 2018).  Diplacone, isolated from Paulownia tomentosa mature fruit, induces ferroptosis-mediated cell death through mitochondrial Ca²⁺ influx and the mitochondrial permeability transition (see TC# 1.A.129) (Oh et al. 2023).

Ca²⁺ flux across the inner mitochondrial membrane (IMM) regulates cellular bioenergetics, intra-cellular cytoplasmic Ca²⁺ signals, and various cell death pathways. Ca²⁺ entry into the mitochondria occurs due to the highly negative membrane potential (ΔΨ_m) through a selective inward rectifying MCU channel (Nemani et al. 2018). In addition to being regulated by various mitochondrial matrix resident proteins such as MICUs, MCUb, MCUR1 and EMRE, the channel is transcriptionally regulated by an upstream Ca²⁺ cascade, post transnational modification and divalent cations. The mode of regulation either inhibits or enhances MCU channel activity and thus regulates mitochondrial metabolism and cell fate (Nemani et al. 2018). MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission (Kadier et al. 2024). Mitochondrial calcium uniporter knockdown in hippocampal neurons alleviates anxious and depressive behavior in the 3XTG Alzheimer's disease mouse model (Wang et al. 2024).

The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein with 2 TMSs. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI (Hicks et al., 2003). Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2 for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium(MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg²⁺ -sensitive ⁴⁵Ca²⁺ uptake by vesicles of an Escherichia coli mutant that is defective in Ca²⁺ and Na⁺ efflux. Hicks et al. (2003) hypothesized that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg²⁺ transporters, Ca²⁺ transporters, probable Mg²⁺/Ca²⁺ channel proteins. Such proteins could provide Mg²⁺ , which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction e.g., during cytoplasmic pH regulation. AtpZ and AtpI have 2 and 4 TMSs respectively.

The Na⁺ F₁F_O ATP synthase of the anaerobic acetogenic bacterium Acetobacterium woodii has a unique hybrid rotor that contains nine copies of a F_O-like c subunit with 1 ion binding site in the 2TMS protein, and one copy of a V_O-like c(1) subunit with one ion binding site in four transmembrane helices. Brandt et al. (2013) cloned and expressed its Na⁺ F₁F_O ATP synthase operon in E. coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli did not substitute for AtpI from A. woodii. Thus, the native AtpI is required for assembly of the hybrid rotor.

The uniporter is membrane potential dependent and sensitive to ruthenium red and its derivative Ru360. It has high conductance and selectivity. Ca²⁺ entry into mitochondria activates the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. MCU is the pore-forming subunit of the uniporter holocomplex. Oxenoid et al. 2016 reported the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore. The channel assembly displays a unique ion channel architecture and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; these rings appear to form the selectivity filter (Oxenoid et al. 2016).

Ca²⁺ uptake into mitochondria regulates bioenergetics, apoptosis, and Ca²⁺ signaling. The primary pathway for mitochondrial Ca²⁺ uptake is MCU. Mitochondrial Ca²⁺ uptake is tightly regulated to maintain low matrix [Ca²⁺] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. Vais et al. 2020 defined a regulatory mechanism in which cytoplasmic Ca²⁺ regulation of intermembrane space-localized MICU1/2 is controlled by Ca²⁺-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca²⁺ that permeates through the channel pore regulates Ca²⁺ affinities of coupled inhibitory and activating sensors in the matrix. Ca²⁺ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca²⁺ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca²⁺ regulation of channel activity. These results demonstrate how Ca²⁺ influx into mitochondria is tuned by coupled Ca²⁺-regulatory mechanisms on both sides of the inner mitochondrial membrane (Vais et al. 2020).

As noted above, MCU controls mitochondrial bioenergetics, and its activity varies greatly between tissues. Xue et al. 2023 highlighted a recently identified MCU-EMRE-UCP1 complex, named thermoporter, in the adaptive thermogenesis of brown adipose tissue (BAT). The thermoporter enhances MCU activity to promote thermogenic metabolism, demonstrating a BAT-specific regulation for MCU activity.

The generalized reaction believed to be catalyzed by (AtpZ)_n, (AtpI)_n and (AtpZ)_n-x AtpIx is:

Mg²⁺ or Ca²⁺ (out) ⇌ Mg²⁺ or Ca²⁺ (in)