Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of adenosine triphosphate (ATP) synthase. Here, we provide evidence that phosphatidylethanolamine (PE) modulates UCP1-dependent proton conductance across the IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to β3 adrenergic receptor agonist–induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PE-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

Oxidative phosphorylation is regulated by mitochondrial calcium (Ca²⁺) in health and disease. In physiological states, Ca²⁺ enters via the mitochondrial Ca²⁺ uniporter and rapidly enhances NADH and ATP production. However, maintaining Ca²⁺ homeostasis is critical: insufficient Ca²⁺ impairs stress adaptation, and Ca²⁺ overload can trigger cell death. In this review, we delve into recent insights further defining the relationship between mitochondrial Ca²⁺ dynamics and oxidative phosphorylation. Our focus is on how such regulation affects cardiac function in health and disease, including heart failure, ischemia-reperfusion, arrhythmias, catecholaminergic polymorphic ventricular tachycardia, mitochondrial cardiomyopathies, Barth syndrome, and Friedreich’s ataxia. Several themes emerge from recent data. First, mitochondrial Ca²⁺ regulation is critical for fuel substrate selection, metabolite import, and matching of ATP supply to demand. Second, mitochondrial Ca²⁺ regulates both the production and response to reactive oxygen species (ROS), and the balance between its pro- and antioxidant effects is key to how it contributes to physiological and pathological states. Third, Ca²⁺ exerts localized effects on the electron transport chain (ETC), not through traditional allosteric mechanisms but rather indirectly. These effects hinge on specific transporters, such as the uniporter or the Na⁺/Ca²⁺ exchanger, and may not be noticeable acutely, contributing differently to phenotypes depending on whether Ca²⁺ transporters are acutely or chronically modified. Perturbations in these novel relationships during disease states may either serve as compensatory mechanisms or exacerbate impairments in oxidative phosphorylation. Consequently, targeting mitochondrial Ca²⁺ holds promise as a therapeutic strategy for a variety of cardiac diseases characterized by contractile failure or arrhythmias.

The mitochondrial calcium uniporter is a multi-subunit Ca²⁺-activated Ca²⁺ channel, made up of the pore-forming MCU protein, a metazoan-specific EMRE subunit, and MICU1/MICU2, which mediate Ca²⁺ activation. It has been established that metazoan MCU requires EMRE binding to conduct Ca²⁺, but how EMRE promotes MCU opening remains unclear. Here, we demonstrate that EMRE controls MCU activity via its transmembrane helix, while using an N-terminal PKP motif to strengthen binding with MCU. Opening of MCU requires hydrophobic interactions mediated by MCU residues near the pore's luminal end. Enhancing these interactions by single mutation allows human MCU to transport Ca²⁺ without EMRE. We further show that EMRE may facilitate MCU opening by stabilizing the open state in a conserved MCU gating mechanism, present also in non-metazoan MCU homologs. These results provide insights into the evolution of the uniporter machinery and elucidate the mechanism underlying the physiologically crucial EMRE-dependent MCU activation process.

Key points: Association of plasma membrane BK_Ca channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK_Ca channel (mitoBK_Ca) by BK‐β subunits is not established. MitoBK_Ca‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBK_Ca display properties similar to that of plasma membrane BK_Ca when associated with BK‐β1 (left‐shifted voltage dependence of activation, V_1/2 = −55 mV, 12 µm matrix Ca²⁺). In BK‐β1 knockout mice, cardiac mitoBK_Ca displayed a low P_o and a depolarized V_1/2 of activation (+47 mV at 12 µm matrix Ca²⁺) Co‐expression of BK_Ca with the BK‐β1 subunit in HeLa cells doubled the density of BK_Ca in mitochondria. The present study supports the view that the cardiac mitoBK_Ca channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBK_Ca shapes mitochondrial Ca²⁺ handling. Association of the plasma membrane BK_Ca channel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK_Ca) with regulatory subunits is unknown. We report that mitoBK_Ca functionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBK_Ca is a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K⁺ of 300 pS. Additionally, mitoBK_Ca displays a high open probability (P_o) and voltage half‐activation (V_1/2 = −55 mV, n = 7) resembling that of plasma membrane BK_Ca when associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK_Ca‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBK_Ca currents (five patches with mitoBK_Ca activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V_1/2 of activation (+47 mV in 12 µm matrix Ca²⁺). The reduced activity of mitoBK_Ca was accompanied by a high expression of BK_Ca transcript in the BK‐β1 KO, suggesting a lower abundance of mitoBK_Ca channels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BK_Ca that specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca²⁺ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK_Ca associates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK_Ca channel that helps to maintain mitochondrial Ca²⁺ homeostasis.

The mitochondrial BK_Ca channel (mitoBK_Ca) is a splice variant of plasma membrane BK_Ca (Maxi-K, BK_Ca, Slo1, K_Ca1.1). While a high-resolution structure of mitoBK_Ca is not available yet, functional and structural studies of the plasma membrane BK_Ca have provided important clues on the gating of the channel by voltage and Ca²⁺, as well as the interaction with auxiliary subunits. To date, we know that the control of expression of mitoBK_Ca, targeting and voltage-sensitivity strongly depends on its association with its regulatory β1-subunit, which overall participate in the control of mitochondrial Ca²⁺-overload in cardiac myocytes. Moreover, novel regulatory mechanisms of mitoBK_Ca such as β-subunits and amyloid-β have recently been proposed. However, major basic questions including how the regulatory BK_Ca-β1-subunit reaches mitochondria and the mechanism through which amyloid-β impairs mitoBK_Ca channel function remain to be addressed.