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Overview

Article One ^[1] Article Two ^[2] Article Three ^[3] Article Four ^[4] Article Five ^[5]

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 eukaryotic life, but activity is regulated differently in each clade.^[2] The precise identity of the MCU wasn't discovered until 2011 and was discovered using a combination of NMR spectroscopy, cryo-electron microscopy, and x-ray crystallography.^[3] Identification of the structure was difficult because it has no apparent sequence similarity to other ion channels.^[2] However, like other ion channels, it is incredibly selective and efficient. The MCU has the ability to only allow 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.^[2]

Under resting conditions, the calcium concentration in the mitochondria is about the same as in the cytoplasm, but when stimulated, it can increase calcium concentration 10-20-fold.^[4] Mitochondria-associated ER membranes (MAMs) exist between mitochondria and the endoplasmic reticulum, the two largest cellular stores of calcium, to allow for efficient transport of calcium ions.^[1] The transfer of electrons through respiratory complexes I-IV produces the energy to pump hydrogen ions into the intermembrane space (IMS) and create the proton electrochemical gradient potential.^[4] This negative electrochemical potential is the driving force that moves positively charged calcium ions into the mitochondrial matrix.^[4]

Regulation of the uptake and efflux of calcium is important to increase calcium levels enough to activate certain enzymes, but also avoid calcium overload and apoptosis.^[1] Mitochondrial calcium increases ATP production by activating pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and isocitrate dehydrogenase in the Krebs cycle.^[1] Therefore, deficiency of MCU leads to decrease of enzyme activity and of oxidative phosphorylation.

Structure

Mitochondrial Calcium Uniporter Complex

The actual mitochondrial calcium uniporter exists as a large complex (around 480 kDa in humans) made up of both pore-forming and regulatory subunits.^[1] The mitochondrial uptake proteins (MICU1 and MICU2) are regulatory proteins in the MCU complex that exist in the IMS and contain EF hand domains for calcium binding to control transport through the channel of the MCU complex.^[1] When calcium ion concentration in the IMS is low, MICU1 and 2 block the MCU to prevent uptake of calcium.^[1] In the presence of high calcium concentrations, more calcium binds to these regulatory proteins and they undergo a conformational change to allow calcium ions through the MCU and into the matrix.^[1] In fact, 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.^[4] Another regulatory protein, MCUR1 is a cofactor in the assembly of the respiratory chain rather than an essential part of the uniporter.^[4] Though the MCU is able to take up calcium independently, there are two other pore-forming subunits, the MCUb and the essential MCU regulator (EMRE).^[1] MCUb is similar to MCU, but certain amino acids differ and make it an inhibitory subunit.^[1] The EMRE is located in the IMS and connects MICU1 and MICU2 to the MCU.^[4] It also contributes to regulation of calcium intake in the MCU.^[1]

Mitochondrial Calcium Uniporter Structure

The MCU was originally thought to be composed of a pentamer of five identical subunits, but it is now known to exist as a dimer of dimers.^[3] More specifically, it is composed of two coiled-coil domains and two transmembrane domains.^[3] The hydrophobic transmembrane domain is located in the inner mitochondrial membrane (IMM) and the hydrophilic coiled-coil domain exists in the mitochondrial matrix.^[2] The transmembrane domain (TMD) consists of eight separate helices (TM1 and TM2 from each subunit) that are connected by mostly hydrophobic amino acids in the IMS and has four-fold symmetry.^[2] TM1 packs tightly against TM2 from the neighboring subunit which conveys a sense of domain-swapping.^[5] This section of the MCU can be roughly divided into a narrow outer leaflet portion with the selectivity filter and lined by the TM2 helices and a wide inner leaflet.^[2] Past the transmembrane domain, the N-terminal domains of the TM1 helices extend into the matrix and form coiled-coils with a C-terminal helix.^[2] These "legs" are separated from each other which allows enough space for calcium ions to diffuse out into the matrix.^[2] Additionally, this domain is responsible for assembly of the MCU and post-translational modification.^[5] Finally, each leg ends in a non-translated domain (NTD).^[2] While the MCU can intake calcium without the NTD, it may have regulatory functions and the ability to bend transmembrane helices to constrict the pore.^{[2] [5]}

Selectivity Filter

Movement of Calcium

Mutations

Medical Relevance

Apoptosis and Cancer

Neurodegenerative Disorders

Diabetes

Regulation and Inhibition

The most well-known and commonly used inhibitor of calcium uptake into the mitochondria is ruthenium red (RuRed). RuRed effectively inhibits calcium uptake without affecting mitochondrial respiration or calcium efflux. Additionally, it has been shown to mitigate tissue damage due to IRI and slow cancer cell migration. The issue with RuRed is that its purification has always been a challenging matter.^[3] Interestingly enough, this led to even more developments in the search for an inhibitor. Many scientists had observed that impure RuRed actually had greater inhibition than pure RuRed. One of the common minor impurities of RuRed, Ru360, was found to be the active component of the RuRed mixtures, meaning it responsible for calcium inhibition. Ru360 is now commercially available and has been widely used for the study of calcium-dependent cellular processes and as a therapeutic agent. Very little is known about its mechanism of inhibtion, but studies show that it interacts with the DXXE motif of the loop connecting the TM1 and TM2 helices.^[3]

Ru360 was a very successful inhibitor, but it showed low cell permeability. So, a new inhibitor called Ru265 was developed which could be easily synthesized and didn't need chromatographic purification. Ru265 had all of the benefits of Ru360, with with twice the cell permeability. Additionally, the same mutations didn't seem to affect it. Mutations of D261 and S259 in human MCU reduced inhibitory effect of Ru360, but not Ru265. Additionally, there were other mutations that affected Ru265, but not Ru360.^[3] This shows how much more research is needed before a mechanism is understood for any inhibitor of the MCU.

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