MitoBK(Ca) is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location.

The large-conductance Ca(2+)- 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 of mitoBK(Ca) as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa 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).