Regulation of the mitochondrial calcium uniporter

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PROJECT SUMMARY Calcium influx into the mitochondria can potently stimulate ATP synthesis, but excessive levels cause mitochondrial failure and cell death. Such calcium overload is a prominent pathological pathway in disease in multiple organ systems. In the heart, this phenomenon is noted during heart attacks, when prolonged ischemia causes calcium to accumulate in the cytoplasm and subsequently overload mitochondria. In heart failure, mitochondrial are also more susceptible to calcium overload. Calcium enters the mitochondria through a multi-subunit calcium-activated channel known as the mitochondrial calcium uniporter. In animal models, genetic inhibition of the uniporter has appeared protective in acute disease. In chronic diseases, though inhibition of calcium overload is protective, there may also be basal requirements for milder mitochondrial calcium uptake. Currently, however, there are no specific therapies to prevent calcium overload or its downstream affects. Pharmacological modulation of the uniporter in vivo is limited by agents that are poorly selective, cell impermeable, or produce off-target effects. A critical gap in the ability to better modulate the uniporter is our limited understanding of how the pore-forming subunit, MCU, is regulated. Recent elegant structural studies have revealed the architecture of the uniporter complex, and mechanisms for calcium selectivity and gating, setting the stage for structure-function investigations of further channel regulation. In this proposal, the principal investigators apply their complementary skills in structural biology and mitochondrial functional assays to define pharmacological and protein-based mechanisms for such channel regulation. First, using a combination of computational, electrophysiological, and structural approaches, we will investigate uniporter inhibitors that are cell-permeable and specific, and useful for either acute or chronic injury. Second, using new molecular tools, mutagenesis, and structural biology, we will identify how the uniporter subunit MCUB leads to inhibition of calcium uptake through the uniporter. Taken together, our studies will reveal novel forms of uniporter regulation that may be developed into therapies for cardiovascular and other disorders.