MitoBK_Ca is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location

The large-conductance Ca²⁺- and voltage-activated K⁺ channel (BK_Ca, MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK_Ca-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK_Ca agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK_Ca (mitoBK_Ca) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK_Ca is the Kcnma1 gene, which produces a protein that migrates at ~140 kDa and arranges in clusters of ~50 nm in purified mitochondria. Physiological experiments further support the origin ofmitoBK_Ca as a Kcnma1 product because NS1619- mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BK _Ca transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK_Ca.