Calmodulin enhancement of mitochondrial calcium uniporter function in isolated mitochondria

Abstract

Mitochondrial calcium (Ca²⁺) uptake and factors that regulate this process have been an area of immense interest given the roles in cellular energetics. Here, we have investigated the ability of the Ca²⁺ sensing protein Calmodulin (CaM) to modify the function of the Mitochondrial Ca²⁺ Uniporter (MCU). Our data leveraged recombinantly produced CaM and mitochondria isolated from healthy and MCU impaired/diseased mice (Barth syndrome model). We found CaM enhanced Ca²⁺ uptake in both the absence and presence of CaMKII inhibition (KN93 as well as AIP). Mitochondria lacking function MCU (Barth syndrome model) validated that MCU was responsible for Ca²⁺ uptake in our experiments. Control experiments demonstrate that the observed CaM enhancement does not arise from CaM Ca²⁺ buffering. Fitting the Ca²⁺fluorescence data supported a monophasic decay process where the presence of CaM yielded enhanced kinetic rates of Ca²⁺ uptake. This CaM enhancement effect persisted in the presence of PTP impairment (cyclosporin), and subtle modification to the CaM protein sequence (D131E) revealed that an intact CaM-C domain Ca²⁺ binding was required for enhancement of MCU function.