MCU Channel Research Articles

Emergence of repurposed drugs as modulators of MCU channel for clinical therapeutics

• The highly selective mitochondrial Ca 2+ uptake channel MCU play a crucial role in cellular Ca 2+ homeostasis and bioenergetics. Recently, De Mario et al., conducted small molecule screen to identify MCU modulators that control mitochondrial Ca 2+ uptake as a proof-of-concept.

Molecular Link between MCU and MRS2P Channels for Mitochondrial Ion Homeostasis and Energy Metabolism

Molecular Link between MCU and MRS2P Channels for Mitochondrial Ion Homeostasis and Energy Metabolism

MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU

MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU

MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU

Ca2+ entry into mitochondria is mediated by the Ca2+uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The roles of MICU proteins are controversial. MICU1 was proposed to be necessary for MCU activity, whereas subsequent studies suggested it inhibits the channel in the low-cytoplasmic Ca2+ ([Ca2+]c) regime, a mechanism referred to as "gatekeeping," that imposes a [Ca2+]c threshold for channel activation at ∼1-3μM. Here, we measured MCU activity over a wide range of quantitatively controlled and recorded [Ca2+]c. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ crosstalk between single inositol 1,4,5-trisphosphate receptor (InsP3R) and MCU channels.

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated inmitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, weadapted inflammatory and hypoxia models. Weidentified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

Pore Architecture and Ion Selectivity Filter of the Mitochondrial Calcium Uniporter

The calcium uniporter of mitochondria is a holocomplex consisting of the calcium-conducting channel, known as the MCU channel, and a score of regulatory proteins. Previous electrophysiology study found that a preeminent property of the uniporter is the high calcium selectivity and conductance and this has been shown to critically depend on the highly conserved amino acid sequence motif, DXXE, in the MCU channel. Our recent NMR data of the MCU channel indicate that the DXXE forms two parallel carboxylate rings at the channel entrance that appear to serve as the ion selectivity filter. We used paramagnetic probes to show that mutants with a single carboxylate ring can bind divalent cation specifically, while in the wild type, the two rings bind the ion cooperatively, resulting in drastically higher apparent affinity. Our new data, together with structural analysis of the DXXE motif, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter of the channel and that Ru360 directly blocks ion entry into the filter.

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.

Abstract 18531: Adrenergic Stimulation Enhances Mitochondrial Ca 2 + Uptake and Cell Death Signaling Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca 2 + Uniporter

Introduction: Mitochondrial Ca 2+ homeostasis is crucial for balancing cell survival and death. The recent breakthrough discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study the role of mitochondrial Ca 2+ regulation in cardiac cells. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca 2+ entry through post-translational modifications of MCU are completely unknown. Hypothesis: Persistent adrenergic signaling induces mitochondrial Ca 2+ overload and activates cell death signaling through MCU tyrosine phosphorylation. Methods: Tyrosine kinase activation and tyrosine phosphorylation of MCU were observed in rat neonatal cardiomyocytes and H9C2 cardiac myoblasts. Mitochondrial Ca 2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca 2+ -sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP, using confocal microscopy. Results: α 1 -adrenoceptor (α 1 -AR) signaling activates mitochondria-localized Ca 2+ -dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, persistent α 1 -AR stimulation increases ROS production, mPTP activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Moreover, pretreatment of a potent Pyk2 inhibitor PF-431396 or overexpression of dominant-negative mutant of MCU effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: The α 1 -AR-Pyk2 dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca 2+ entry and apoptosis in cardiac cells. Pyk2-MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca 2+ overload, oxidative stress and cardiac injury under pathophysiological states such as heart failure, where catecholamine levels highly increase.

Identification and Analysis of Cation Channel Homologues in Human Pathogenic Fungi

Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K+), calcium (Ca2+) and transient receptor potential (Trp) channels, but not sodium (Na+) channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated Kv channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca2+ uniporter (MCU). In contrast to humans, which express many K+, Ca2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K+, Ca2+ and Trp channel homologues. Furthermore, the sequences of fungal K+, Ca2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

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

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.

Patch-Clamp Analysis of Mitochondrial Ca2+ Uniporter in Different Tissues

The mitochondrial Ca2+ uptake has been recognized as a central player in cellular pathophysiology for decades. Pathways of Ca2+ transport across the inner mitochondrial membrane (IMM) were investigated indirectly with biochemical techniques and most recently by applying the patch clamp technique to mitoplasts isolated from mammalian systems. Patch-clamp recording of the IMM have proved to provide an unambiguous picture of mitochondrial Ca2+ uptake under strictly controlled conditions. In this study, we evaluated and compared the biophysical properties of mitochondrial Ca2+ uptake in different mouse tissues. Freshly isolated mitoplasts from mouse heart, skeletal muscle, liver, kidney, spleen and brown adipose tissue were patch clamped in the whole-mitoplast configuration. Voltage step and ramp protocols covering the whole range of physiological potentials were applied to elicit the inwardly rectifying Ca2+current sensitive to RuR known as MCU. The distribution of MCU current densities between tissues was as follows: spleen > brown adipose tissue ≈ skeletal muscle > kidney > liver >> heart. Interestingly, MCU current density in heart was about 10 times smaller than in skeletal muscle. Our results support the view of a differential tissue activity of MCU, which can be explained by either a different distribution of MCU channel and/or by a different expression of its regulatory subunits confering various modes of physiological regulation. Further on, in order to investigate the putative direct contribution of Uncoupling Protein Isoform 2 and 3 (UCP2/3) to the uniporter, we investigated MCU properties in wild type (WT) mouse tissues where these two proteins have been found to have a major role and compared its properties in the correspondent tissues of UCP2 and UCP3 KO mice. We found no significant differences in calcium currents of any of the WT tissues studied compared to their UCP2/3 KO counterparts.

Cd(2+)-induced cytochrome c release in apoptotic proximal tubule cells: role of mitochondrial permeability transition pore and Ca(2+) uniporter.

Cd(2+) induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound-induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd(2+)-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 muM Cd(2+) induced approximately 5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosis-inducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd(2+) concentrations at 24 h, suggesting that elevated Cd(2+) may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd(2+) caused swelling of energized isolated kidney cortex mitochondria (EC(50) approximately 9 muM) and cytochrome c release, which were independent of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd(2+) inhibited swelling and cytochrome c release induced by PTP openers (PO(4)(3-) or H(2)O(2)+Ca(2+)). The mitochondrial Ca(2+) uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La(3+), ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd(2+) inhibition of PO(4)(3-)-induced swelling. Using the Cd(2+)-sensitive fluorescent indicator FluoZin-1, Cd(2+) was also taken up by mitoplasts. The aquaporin inhibitor AgNO(3) abolished Cd(2+)-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd(2+) concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane.