The subproteome of mitoBKCa channels from cardiomyocytes reveals novel insights into its mitochondrial import mechanism and function.

摘要

BKCa channels are widely expressed ion channels and characterized by their large conductance to potassium and sensitivity to calcium and voltage. It is typically observed at the plasma membrane in the majority of cell types, with adult cardiomyocytes being an exception. Previously, both electrophysiological and immunological studies have detected BKCa channels in cardiomyocytes mitoplasts, confirming the presence of this K+ channel (mitoBKCa); however, its physiological functions have not yet fully understood.;This work is the first one to examine the interactome of mitoBK Ca channels in adult heart (isolated cardiomyocytes and whole ventricle). We used a directed proteomic approach aided by co-immunoprecipitation with BKCa antibodies and pull-down with recombinant DEC sequences. As a result, we identified an extensive network of the mitoBKCa, channel and overall, a total of 1079 different proteins were identified as partners of the BKCa channel in cardiomyocytes and left ventricle, including 151 mitochondrial proteins. Two putative protein partners were selected to validate and further examine the associations with BKCa channels: i) the Tom22 from the mitochondrial import system, and ii) the adenine nucleotide translocator (ANT), which is linked to oxidative phosphorylation and the regulation of mPTP.;We first demonstrated the interactions of mitoBKCa with Tom22 through reciprocal co-immunoprecipitation (CO-IP), and then further confirmed that both Tom22 and BKCa were targeted into mitochondria through cell fractionation. This result raises the possibility of the functional interaction between the two proteins in mitochondria, supporting a potential import mechanism of BKCa channel through Tom22. Additionally, we identified the transmembrane domain of BKCa channel (1-711) as the main interacting regions with Tom22 through CO-IP. Next, we verified and studied the interactions between mitoBKCa and ANT with similar approaches. As a result, mitoBKCa was able to co-immunoprecipitate ANT, and the presence of DEC sequence in BKCa-DEC enhanced the ability of ANT to associate with BKCa by ~30%. More importantly, this interaction has a very high likelihood to happen in the mitochondria, as supported by the cell fractionation experiment that the majority of ANT and a considerate amount of mitoBK Ca were targeted into mitochondria. This finding indicates a molecular link between mitoBKCa and mPTP, as ANT acts like an important regulatory component of it. Furthermore, the transmembrane domain of BKCa (1-343) can also contribute to the molecular interaction between BKCa channel and ANT.