Objective: Mitochondrial calcium-activated K + (mitoK Ca ) channels have been described as channels that are activated by Ca 2+ , inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK Ca channels. Here, we show several weaknesses in this hypothesis. Methods: Isolated mitochondria from rat hearts were tested for evidence of mitoK Ca activity by analyzing functional

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... s in K + -rich and K + -free media. Results: NS-1619 promoted mitochondrial depolarization both in K + -rich and K + -free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK Ca channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K + in the media was substituted with tetraethylammonium (TEA + ), which is not transported by any known K + carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K + . In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK Ca activity by altering Ca 2+ concentrations in the medium and inducing mitochondrial uncoupling. Conclusion: Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K + transport in isolated heart mitochondria compatible with mitoK Ca opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK Ca and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.