Large conductance Ca²⁺-activated potassium channels (BK_Ca channels) are widely expressed in plasma membrane of various cell types. Interestingly, recent studies provided the evidence for the existence of BK_Ca channels also in mitochondria. However, the molecular composition of these channels as well as their cellular and tissue distribution is still unknown. Hence, the goal of the present study was to search for the potential candidate for the regulatory component of neuronal mitoBK_Ca channel. A combined approach of Western Blot analysis, immunofluorescence and electron microscopy with the use of antibodies directed against four distinct betasubunits demonstrated the presence of BK_Ca channel beta4 subunit (KCNMB4) in the inner membrane of neuronal mitochondria. Double-labeling immunocytochemistry in paraffin embedded rat brain sections as well as in primary culture of rat hippocampal dentate gyrus revealed the colocalization of BK_Ca channel beta4 subunit with mitochondrial staining. Furthermore, the distribution pattern of beta4 subunit indicated that its expression is restricted to the subpopulation of mitochondria within the cell. Electron microscopy data additionally confirmed mitochondrial residence of beta4 and proved its close association with inner mitochondrial membrane. These results therefore suggest that beta4 subunit could be a potential candidate for the regulatory component of mitochondrial BK_Ca channels in neurons.

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