Abstract 570: Mechanism Of Mitochondrial Calcium Uniporter Regulation

• Published in: Circulation research Vol. 125; no. Suppl_1
• Main Authors: Garg, Vivek, Paranjpe, Ishan, Unsulangi, Tiffany, Suzuki, Junji, Milescu, Lorin S, Kirichok, Yuriy
• Format: Journal Article
• Language: English
• Published: 02.08.2019
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/res.125.suppl_1.570

Abstract only Calcium uptake by mitochondria plays an important role in sequestering cytosolic calcium, regulating ATP production and maintaining the redox homeostasis. Mitochondrial calcium uniporter (MCU), a macromolecular channel complex that resides in the inner mitochondrial membrane, is the primary electrogenic pathway for calcium influx into the matrix. Because calcium uptake by mitochondria is dictated by the kinetic equilibrium between the influx and efflux machinery, the patch clamp is the best method to measure electrogenic MCU activity in isolation from the efflux mechanisms. Here we report the development of a system for heterologous expression of MCU and its associated subunits, essential MCU regulator (EMRE), MCUb and MICU1–3. Using this system, we investigated the biophysical properties of the MCU pore and its regulation. The system is based on a recombinant cell line (Drp1 -/- mouse embryonic fibroblasts) in which knockouts for all subunits of the MCU complex have been generated. This system allowed both optical and direct electrophysiological studies of the structure-function relationships in the MCU complex with a relative ease in comparison to other cell lines. We confirmed the essential role of pore-forming MCU and EMRE subunits in mediating the MCU current. Importantly, fast-step solution exchange and single-channel recordings allowed us to gain novel insights into the biophysical properties of MCU e.g. inward rectification, kinetics of activation and selectivity to monovalent vs divalent ions.