User:R. Jeremy Johnson/Mitochondrial Calcium Uniporter

Overview

The mitochondrial calcium uniporter (MCU) complex is the main source of entry for calcium ions into the mitochondrial matrix from the intermembrane space. MCU channels exist in most eukaryotes, but activity is regulated differently in each clade.^[1] MCU was definitively assigned in 2011 using a combination of NMR spectroscopy, cryoelectron microscopy, and x-ray crystallography.^[2] Recent cryoelectron microscopy (cryo-EM) analysis provides a structural framework for understanding the mechanism for calcium selectivity by the MCU.^[3] Like other ion channels, the MCU is highly selective and efficient, allowing calcium ions into the mitochondrial matrix at a rate of 5,000,000 ions per second, even though potassium ions are over 100,000 times more abundant in the intermembrane space.^[1]

Under resting conditions, the calcium concentration in the mitochondria is about the same as in the cytoplasm, but when stimulated, mitochondrial calcium concentration increases 10 to 20-fold.^[3] Mitochondria-associated ER membranes exist between the mitochondria and the endoplasmic reticulum facilitate efficient transport of calcium ions.^[4] The transfer of electrons through respiratory complexes I-IV produces the energy to pump hydrogen ions into the intermembrane space and establish the proton electrochemical gradient potential.^[3] This negative electrochemical potential is the driving force that moves positively charged calcium ions into the mitochondrial matrix.^[3] Calcium uptake and efflux must be tightly regulated to controll essential Krebs cycle enzyme activity, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase, while avoiding calcium overload and apoptosis.^[4]

Mitochondrial Calcium Uniporter Complex

The MCU complex exists as a large complex (around 480 kDa in humans) made up of both pore-forming and regulatory subunits.^[4] The MCU complex is composed of the MCU as well as regulatory subunits including the mitochondrial calcium uptake proteins MICU1 and MICU2, essential MICU regulator (EMRE), MCU regulatory subunit b (MCUb), and MCU regulator 1 (MCUR1). ^[5] The mitochondrial uptake proteins (MICU1 and MICU2) are intermembrane regulatory proteins that use their EF hand domains to grab intermembrane calcium and to control transport through the channel of the MCU.^[4] When calcium ion concentration in the intermembrane space is low, MICU1 and 2 block the MCU to prevent uptake of calcium.^[4] In high calcium concentrations, calcium binds to these regulatory proteins and they undergo a conformational change to allow calcium ions through the MCU and into the matrix.^[4] When calcium levels are below 500 nM, MICU1 can block movement of calcium by itself, calcium levels between 500 nM and 1,500 nM require both MICU1 and MICU2 to block ion entry, and any concentration over 1,500 nM is sufficient for calcium entry.^[3] Another regulatory protein, MCUR1 is a cofactor in the assembly of the respiratory chain rather than an essential part of the uniporter.^[3] Though the MCU is able to take up calcium independently, two other membrane spanning subunits, the MCUb and the essential MCU regulator (EMRE), connect to the MCI and add further regulatory mechanisms.^[4] MCUb is similar to MCU, but through key amino acid substitutions serves an inhibitory role.^[4] The EMRE contributes to regulation of calcium intake in the by connecting MICU1 and MICU2 to the MCU.^[3][4]

Figure 1: Structure of mitochondrial calcium uniporter colored by functional domain. The transmembrane domain is highlighted in salmon, the linker domain spanning the mitochondrial matrix in light cyan, coiled-coil domain in dark violet, and the N-terminal domain in slate blue. PDB 6DT0

MCU Structure

The MCU is the ion channel component of the MCU complex (Figure 1). An NMR structure of an inactive MCU from C. elegans showed a pentameric arrangement, but the recent crystal and cryo-EM structures of multiple MCUs reaffirmed that active eukaryotic MCU exists as four monomers, identical in sequence, arranged and packed together such that they structurally form a dimer of dimers in a tetrameric truncated pyramid (Figure 2).^[2] The MCU protein is composed of a transmembrane domain, a coiled coil domain, and a N-terminal domain (NTD) (Figure 1).^[2] The hydrophobic transmembrane domain is located in the (inner mitochondrial membrane) while the hydrophilic coiled-coil domain and NTD are positioned in the mitochondrial matrix.^[1]

Figure 2: Symmetry and organization of MCU dimer of dimers viewed from the inner mitochondrial membrane PDB 6DT0

Transmembrane Domain

The transmembrane domain is inserted into the inner mitochondrial membrane and opens to the inner membrane space (Figure 1). The transmembrane domain consists of eight separate helices (TM1 and TM2 from each subunit) with four-fold symmetry that are connected by hydrophobic amino acids in the intermembrane space (Figure 2).^[1] The small selectivity filter pore, highly specific for calcium binding is located in the interior of the bundle of four packed TM2 helices while the four helices of TM1 surround this TM2 bundle. TM1 packs tightly against TM2 from the neighboring subunit which conveys a sense of domain-swapping.^[5]

Coiled-coil Domain

The N-terminal domains of the TM1 helices extend into the matrix and form coiled-coils with a C-terminal helix.^[1] These "legs" are separated from each other which allows enough space for calcium ions to diffuse out into the matrix.^[1] The coiled coil domain is the first subsection of the soluble domain, which resides in the inner mitochondrial membrane. The coiled coil functions as the joints of the uniporter, providing flexibility to promote transport of Ca²⁺ions down their concentration gradient.^[5] When Ca²⁺ ions binds to the selectivity pore, the coiled-coil swings approximately 8° around its end near the NTD. This movement propagates to the top of the transmembrane domain, where the pore is located about 85 Å away. The coiled coil's flexibility can be attributed to the disordered packing between subunits. Subunits A and C adopt different conformations than the B and D subunits, although they superimpose closely.^[5] The coiled-coil domain is also required for proper assembly of the MCU and is post-translationally modified.^[5]

N-terminal Domain

Each leg of the coiled-coil domain extends into the NTD.^[1] The soluble domain, including the linker, coiled-coil, and NTD, is wider than the transmembrane domain (Figure 1), allowing calcium ions passed through the selectivity filter to rehydrate, increasing the conductivity of ions through the uniporter into the mitochondrial matrix.^[5] While the MCU can intake calcium without the NTD, the NTD serves regulatory functions, including bending transmembrane helices to constrict the pore.^[1][5] Reorganization in the NTD due to shifts in the coiled coil domain alters membrane packing, facilitating a rotamer switch between a pair of tyrosine residues controlling calcium flow through the pore.

Selectivity Filter

Figure 3: Electrostatic potential of MCU viewed from the intermembrane space. The high concentration of negatively charged residues (red), surrounding the selectivity filter, attracts the positively charged calcium ions PDB 6DT0

The selectivity filter of the MCU is composed of multiple layers of acidic amino acids near the narrow mouth of the channel and is responsible for the high affinity and selectivity of the MCU for calcium (dissociation constant of less than 2nM) (Figure 3).^[1] Negatively charged aspartates (Asp333) at the mouth of the MCU congregate positively charged calcium ions at the entrance of the channel.^[1] A highly conserved WDXXEP motif in the TM2 helices form the selectivity pore which selects for calcium transport over other similar ions.^[1]

The WDXXEP motif consists of Trp332 at the N-terminal end, Asp333, Glu336, and Pro337.^[1] The negatively charged side chains of Asp333 and Glu336 point towards the pore.^[1] The diameter of the pore created by the carboxyl ring on the 4 identical glutamates (Glu336) is about 4Å, allowing only a dehydrated Ca²⁺ ion to bind. The combination of these radii and high negative charge (Figure 3) account for the selectivity of the MCU. For example, potassium has an ionic radius of 1.38Å which is much larger than the 1.00Å ionic radius of calcium and thus cannot fit through the pore.^[1] Additionally, even though sodium ions have a similar ionic radius, the +2 charge on calcium is better matched for coordination with the glutamate residues.^[1]

The additional residues of the WDXXEP motif, Trp332 and Pro337 pack against each other, are oriented towards the pore, and serve to stabilize Asp333 and Glu336.^[1][5] Trp332 stabilizes the carbonyl side chains of Glu336 through hydrogen bonding and anion pi interactions. Approximately one helical turn below the glutamate ring of the selectivity filter, a wider tyrosine ring (12Å) facilitates calcium rehydration after passage through the selectivity pore.^[5]

Movement of Calcium

Cryo-EM showed three calciums in the MCU channel of roughly spherical density equally spaced 6Å apart.^[1] Sites 1 and 2 lie within the selectivity filter and likely contain calcium, but site 3 could be calcium or some other small molecule.^[1] Site 1 is positioned in the ring formed by Asp333 residues with a distance of 8Å between the center of the site and each carboxylate group indicating the presence of water.^[1] Site 2 is positioned in the ring formed by Glu336 with a smaller distance (4.0Å) between the carboxylate group of each residue and the middle of the site, indicating the absence of water.^[1] For transporting calcium, a mechanism has been proposed where one calcium ion coordinated with water positioned in site 1 is dehydrated and moves to site 2 while a new calcium ion moves from the intermembrane space into site 1.^[1] Meanwhile, a different calcium ion moves from site 2 to site 3 and becomes rehydrated upon passage into the mitochondrial matrix.^[1]

Mutations

A number of mutations completely eliminate calcium uptake by the MCU. For example, mutation of an residue in the WDXXEP motif, with the exception of the two "X" residues, altered the highly conserved selectivity filter and completely eliminated calcium uptake.^[1][5] Even substituting Glu336 with an aspartate residue significantly changes the dimensions of the pore and inhibits uptake of calcium. Mutation of the secondary tyrosine ring substantially impaired calcium intake and proper protein folding.^[5] Additional mutations outside the selectivity filter also impacted calcium uptake, including Trp317 (analogous to Trp210 in C. europaea) which has a side chain constituting a primary contact point between TM1 and TM2.^[5] Mutation of human MCU Phe326 (analogous to Phe218 in C. europaea) or Gly331 of the TM1-TM2 linker (Gly223 in C. europaea) also affected the linker conformation and configuration of the pore entrance and impaired calcium intake.^[5]

Regulation and Inhibition

Figure 4: Structures of the ruthenium-based inhibitors of the MCU. Created using ChemDraw Professional 16.0

The most well-known and commonly used inhibitor of the MCU is ruthenium red (RuRed).^[2] RuRed is a trinuclear, oxo-bridged complex that effectively inhibits calcium uptake without affecting mitochondrial respiration or calcium efflux. The disadvantage of ruthenium red is its challenging purification.^[2] Interestingly, a compound identified in an impure version of RuRed, termed Ru360, was found to be the actual active component as an inhibitor of the MCU (Figure 4). Ru360 is a binuclear, oxo-bridged complex with a similar structure to that of RuRed. To increase the cell permeability of Ru360, the derivative Ru265 was subsequently which had twice the cell permeability of Ru360. Ru265 possesses two bridged Ru centers bridged by a nitride ligand (Figure 4).^[2]

Recent experiments suggest that Ru360 inhibits calcium uptake through interactions with the WDXXEP motif. However, not much is known about the mode of inhibition. Also, mutations of Asp261 and Ser259 in human MCU were shown to maintain calcium uptake into the matrix, but reduce the inhibitory effect of Ru360, but not Ru265. However, a mutation in a cysteine residue in the NTD had the opposite effect as it reduced the inhibitory effects of Ru265, but not Ru360 (Figure 4).^[2]

Medical Relevance

The MCU is connected with various diseases due to its effect on apoptosis and cell signaling. The overload of the mitochondrial matrix with calcium leads to release of cytochrome c, overproduction of reactive oxygen species, mitochondrial swelling, and the opening of the mitochondrial permeability transition pore (mPTP) which all lead to apoptotic cell death.^[2] This connection between mitochondrial calcium and apoptosis makes MCU dysregulation a large contributor to cell death and disease. Calcium machinery in the mitochondria are targets for proto-oncogenes and tumor suppressors for this very reason.^[3] Apoptosis can either be induced or repressed. Furthermore, external stimuli can activate receptors in the endoplasmic reticulum that release calcium and activate signal transductions.^[4] Sequestration of calcium in the mitochondria is vital to shut down these activations, so any impact in movement of calcium ions can cause a wide variety of diseases.^[4]

Neurodegenerative Disorders

Disruption in calcium homeostasis leads to a wide range of neurodegenerative disorders. The MCU complex plays a role in neuromuscular disease because of a loss of function of the MICU1 subunit.^[2] Mutation of MICU1 causes myopathy, learning difficulties, and progressive movement disorders which can be lethal. In Alzheimer's disease, the buildup of amyloid-β plaques in the brain leads to increased calcium uptake in neurons and cell death. Similarly, in early onset Parkinson's disease, degradation of MICU1 by the ligase Parkin leads to increased mitochondrial calcium uptake, overload, and death. Finally, disrupted glutamate homeostasis in astrocytes and neurons leads to calcium overload and cell death via excitotoxicity in Amyotrophic Lateral Sclerosis (ALS).^[2]

Diabetes

Calcium homeostasis misregulation is also instrumental in obesity, insulin resistance, and type-II diabetes.^[4] The intracellular calcium concentrations in primary adipocytes from obese human subjects are elevated. Any inhibition of downstream calcium signaling could decrease movement of the GLUT4 glucose transporter and glucose uptake. Additionally, removal of MCU in β-cells in the pancreas demonstrated a decrease in cellular ATP concentration following glucose stimulation which resulted in decreased glucose-stimulated insulin secretion.

Heart Failure

Calcium overload in the mitochondria of cardiac cells lead to apoptotic cardiac cell death. Calcium governs excitation contraction coupling of the cardiac muscles, which creates the ATP needed to power the contraction during heart beats. The increase in mitochondrial Ca²⁺ concentration is essential for the functioning of this muscle contraction. Mitochondrial Ca²⁺ overload, though, leads to necrotic cardiac cell death and can be targeted with regulation of the MCU. An example of potential treatment might involve the use of Ru360 to inhibit the uptake of Ca²⁺ ions into the mitochondria.^[3]