Nora Eccles Harrison Cardiovascular Research & Training Institute, University of Utah, Salt Lake City, UT, USA.
Department of Anesthesiology and Perioperative Medicine, Division of Molecular Medicine.
J Physiol. 2019 Aug;597(15):3817-3832. doi: 10.1113/JP277769. Epub 2019 Jul 11.
Association of plasma membrane BK channels with BK-β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK channel (mitoBK ) by BK-β subunits is not established. MitoBK -α and the regulatory BK-β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBK display properties similar to that of plasma membrane BK when associated with BK-β1 (left-shifted voltage dependence of activation, V = -55 mV, 12 µm matrix Ca ). In BK-β1 knockout mice, cardiac mitoBK displayed a low P and a depolarized V of activation (+47 mV at 12 µm matrix Ca ) Co-expression of BK with the BK-β1 subunit in HeLa cells doubled the density of BK in mitochondria. The present study supports the view that the cardiac mitoBK channel is functionally modulated by the BK-β1 subunit; proper targeting and activation of mitoBK shapes mitochondrial Ca handling.
Association of the plasma membrane BK channel with auxiliary BK-β1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK ) with regulatory subunits is unknown. We report that mitoBK functionally associates with its regulatory subunit BK-β1 in adult rodent cardiomyocytes. Cardiac mitoBK is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K of 300 pS. Additionally, mitoBK displays a high open probability (P ) and voltage half-activation (V = -55 mV, n = 7) resembling that of plasma membrane BK when associated with its regulatory BK-β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK -α and its BK-β1 subunit. Mitochondria from the BK-β1 knockout (KO) mice showed sparse mitoBK currents (five patches with mitoBK activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V of activation (+47 mV in 12 µm matrix Ca ). The reduced activity of mitoBK was accompanied by a high expression of BK transcript in the BK-β1 KO, suggesting a lower abundance of mitoBK channels in this genotype. Accordingly, BK-β1subunit increased the localization of BKDEC (i.e. the splice variant of BK that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-β1 KO mitochondria displayed a more rapid Ca overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK associates with its regulatory BK-β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK channel that helps to maintain mitochondrial Ca homeostasis.
质膜 BK 通道与 BK-β 亚基的结合改变了其生物物理特性和生理作用；然而，BK-β 亚基对线粒体 BK 通道（mitoBK）的功能调节尚未确定。小鼠心脏线粒体中存在 BK-α 和调节性 BK-β1 亚基的相互作用。当与 BK-β1 结合时，相当一部分 mitoBK 表现出类似于质膜 BK 的特性（激活的电压依赖性左移，V=-55 mV，基质 Ca 为 12 µm）。在 BK-β1 敲除小鼠中，心脏 mitoBK 的 P 较低，激活的 V 去极化（在基质 Ca 为 12 µm 时为+47 mV）。HeLa 细胞中 BK 与 BK-β1 亚基的共表达使线粒体中 BK 的密度增加了一倍。本研究支持这样的观点，即心脏 mitoBK 通道受 BK-β1 亚基的功能调节；mitoBK 的适当靶向和激活塑造了线粒体 Ca 处理。
质膜 BK 通道与辅助性 BK-β1-4 亚基的结合，深刻影响着该通道参与的调节机制和生理过程。然而，线粒体 BK（mitoBK）与调节亚基的功能联系尚不清楚。我们报道，在成年啮齿动物心肌细胞中，mitoBK 与调节性亚基 BK-β1 具有功能相关性。心脏 mitoBK 是一种钙激活电压门控通道，对巴氯芬敏感，电导为 300 pS。此外，mitoBK 显示出高开放概率（P）和电压半激活（V=-55 mV，n=7），类似于与调节性 BK-β1 亚基结合时的质膜 BK。免疫化学测定显示线粒体 BK-α与 BK-β1 亚基之间存在相互作用。来自 BK-β1 敲除（KO）小鼠的线粒体显示稀疏的 mitoBK 电流（28 个总斑中只有 5 个有 mitoBK 活性），激活的 V 去极化（在基质 Ca 为 12 µm 时为+47 mV）。mitoBK 活性降低伴随着 BK-β1 KO 中 BK 转录本的高表达，表明该基因型中 mitoBK 通道的丰度较低。相应地，BK-β1 亚基将 BKDEC（即特异性靶向线粒体的 BK 剪接变体）的定位增加到线粒体中二倍。重要的是，巴氯芬处理和 BK-β1 KO 线粒体都显示出更快的 Ca 过载，表现为线粒体通透性转换孔的早期开放。我们提供了强有力的证据表明，mitoBK 与心脏线粒体中的调节性 BK-β1 亚基结合，确保了 mitoBK 通道的适当靶向和激活，有助于维持线粒体 Ca 稳态。
