BK Channel Subproteome Includes Novel Mitochondrial Partners: ADP/ATP Carrier and TOM 22 Import Receptor

Abstract

BK channels are large conductance voltage- and Ca2+-sensitive potassium-selective channels. A specific splice variant, BK-DEC (mitoBKCa), is targeted to mitochondria of adult cardiomyocytes. MitoBKCa activation protects the heart from ischemia and reperfusion injury. To understand its physiological relevance in the heart we characterized the mitoBKCa-associated subproteome in rat cardiomyocytes.Three strategies were implemented: 1) Pulldown of DEC-interacting cellular proteins from left ventriculocytes using recombinant GST-DEC 2) Pulldown of DEC-interacting mitochondrial proteins from isolated left ventriculocytes mitochondria using recombinant GST-DEC and 3) Immunoprecipitation of mitoBKCa-interacting mitochondrial proteins with BK monoclonal antibody and control IgG; mitochondrial proteins were purified from left ventricles. Protein composition was analyzed with 1-D-SDS-PAGE, followed by in-gel trypsinization and LC/MS/MS Mass Spectrometry. These approaches provide a detailed view of mitoBKCa associated subproteome in cardiomyocytes.We identified 516 interacting partners with mitoBKCa with a score>21, including 161 mitochondrial proteins. Gene ontology analysis of mitoBKCa-associated mitochondrial subproteome revealed mitoBKCa association with 13 distinct mitochondrial functions in the heart. For example, DEC interacts with ADP/ATP translocase 1 (AAC) (2 peptides, n=1, score=43), suggesting a role in energy balance. Additionally, DEC also associates with TOM22 (1 peptide, n=1, score=45), suggesting a BK mitochondrial import mechanism through the TOM system. We verified interactions of mitoBKCa with TOM22 and AAC by expressing BK and TOM22 or AAC in HEK and detecting their interactions with reciprocal coimmunoprecipitation. Second, we demonstrated that BK channel, TOM22 and AAC are targeted into mitochondria using cell fractionation and confocal microscope imaging. In conclusion, proteomic analysis identified a wide variety of cellular and mitochondrial proteins forming complex with mitoBKCa channels. BK association with AAC and TOM22 suggests its involvement in regulating mitochondrial energetic balance, and a TOM22 mediated mechanism for mitochondrial targeting.