The Subproteome of mitoBKCa from Cardiomyocytes Reveals Novel Insights into BK Channel Function and Pathology

The mitochondrial large-conductance Ca2+ -activated K+ channel (mitoBKCa) of adult cardiomyocytes contains a C-terminal 50 amino acid insert (DEC) as essential for mitochondrial targeting. The activation of mitochondrial BK channels (mitoBKCa) protects the heart from ischemia and reperfusion injury, highlighting its significant role in cardiac function. Here, we characterized the subproteome of mitoBKCa in cardiomyocytes in the quest to better understand its physiological and pathological relevance in the heart. To this end, we used GST-DEC and GST proteins to pull down proteins interacting with DEC using cardiomyocyte lysates and mitochondrial lysates. We also used monoclonal BK antibody and IgG to immunoprecipitate proteins interacting with mitoBKCa using Percoll purified mitochondrial lysates from whole heart. In all, we identified 212 cellular proteins and 157 mitochondrial proteins interacting with DEC; and 60 mitochondrial proteins interacting with mitoBKCa. Gene oncology analysis revealed mitoBKCa potential contribution to multiple cellular functions. It could contribute to oxidative respiration through cytochrome c oxidase (5 subunits) and ADP/ATP translocase (AAC), including AAC1 (10 peptides, n=2) and AAC2 (13 peptides, n=2), and regulate mitochondrial metabolism through acetyl-Coenzyme transferase 2 (7 peptides, n=2). In addition, our data revealed that mitoBKCa has a broader impact. It might facilitate the pathological process of Parkinson's disease and multiple types of cancer by interacting with critical enzymes, such as NADH dehydrogenase (7 subunits, 3-7 peptides each) and integrin linked kinase (2 peptides, n =1). Excitingly, mitoBKCa interacted with 15 different subunits of the ATP synthase, which forms the permeability transition pore, underscoring a new mechanism for mitoBKCa to protect the heart. In summary, proteomic analysis identified a wide variety of cellular and mitochondrial proteins forming complex with mitoBKCa channel, revealing new paradigms for BK channel function.