MitoBKCa channel is functionally associated with its regulatory β1 subunit in cardiac mitochondria

Key points Association of plasma membrane BKCa channels with BK-beta subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBK(Ca)) by BK-beta subunits is not established. MitoBK(Ca)-alpha and the regulatory BK-beta 1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBK(Ca) display properties similar to that of plasma membrane BKCa when associated with BK-beta 1 (left-shifted voltage dependence of activation, V-1/2 = -55 mV, 12 mu m matrix Ca2+). In BK-beta 1 knockout mice, cardiac mitoBK(Ca) displayed a low P-o and a depolarized V-1/2 of activation (+47 mV at 12 mu m matrix Ca2+) Co-expression of BKCa with the BK-beta 1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBK(Ca) channel is functionally modulated by the BK-beta 1 subunit; proper targeting and activation of mitoBK(Ca) shapes mitochondrial Ca2+ handling. Association of the plasma membrane BKCa channel with auxiliary BK-beta 1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK(Ca)) with regulatory subunits is unknown. We report that mitoBK(Ca) functionally associates with its regulatory subunit BK-beta 1 in adult rodent cardiomyocytes. Cardiac mitoBK(Ca) is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBK(Ca) displays a high open probability (P-o) and voltage half-activation (V-1/2 = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-beta 1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa-alpha and its BK-beta 1 subunit. Mitochondria from the BK-beta 1 knockout (KO) mice showed sparse mitoBK(Ca) currents (five patches with mitoBK(Ca) activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V-1/2 of activation (+47 mV in 12 mu m matrix Ca2+). The reduced activity of mitoBK(Ca) was accompanied by a high expression of BKCa transcript in the BK-beta 1 KO, suggesting a lower abundance of mitoBK(Ca) channels in this genotype. Accordingly, BK-beta 1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-beta 1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK(Ca) associates with its regulatory BK-beta 1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK(Ca) channel that helps to maintain mitochondrial Ca2+ homeostasis.