Pharmacological and physiological stimuli do not promote Ca2+-sensitive K+ channel activity in isolated heart mitochondria

Objective: Mitochondrial calcium-activated K+ (mitoK(Ca)) channels have been described as channels that are activated by Ca2+, 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 mitoKCa activity by analyzing functional parameters 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 Ca2+ 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. C) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.