Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter

Mitochondrial calcium homeostasis is vital to cellular activities such as aerobic ATP production and cell death. Calcium uptake by mitochondria is achieved using an intricately regulated calcium channel, known as mitochondrial calcium uniporter (MCU). MCU demonstrates high calcium selectivity and conductance but little is known about its ion selectivity filter. We used paramagnetic NMR to show that the double carboxylate rings formed by the "DXXE" signature sequence of MCU is the ion selectivity filter and that the two ion binding sites can afford high ion affinity via positive cooperativity. We also showed that the channel inhibitor Ru360 directly blocks at the selectivity filter. Our approach demonstrates the promising application of NMR in investigating ion channels interacting with ions and blockers.