MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress
MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress
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DOI：
10.1161/circulationaha.118.037968
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发表时间：
2019-11-19
期刊：
影响因子：
37.8
通讯作者：
Elrod, John W.
中科院分区：
文献类型：
--
作者：
Lambert, Jonathan P.;Luongo, Timothy S.;Elrod, John W.
背景：线粒体钙单转运体(MTCU)是一种位于线粒体内膜上的约700-kD的多亚单位通道，是线粒体摄取钙(Ca-m(2+))所必需的。在这里，我们详细描述了MCUB在MTCU调节和功能中的作用，并首次研究了MCUB与心脏生理学的相关性。方法：利用CRISPR-Cas9n技术建立了稳定的MCUB基因敲除细胞系(MCUB-/-)，并建立了心脏特异的、三苯氧胺诱导的MCUB突变小鼠(CAG-CAT-MCUB x MCM；MCUB-TG)，用于在体评价心脏生理和对缺血/再灌注损伤的反应。用活细胞成像和高分辨荧光分光光度法检测细胞内钙离子交换和大小排斥层析；用蓝色天然PAGE和免疫沉淀检测MCUB的分子功能和对高分子量MTCU复合体的影响。结果：利用遗传功能获得和功能丧失的方法，我们发现MCUB的表达取代了功能MTCU复合体中的MCU，从而减少了线粒体钙摄取1和2(MICU1/2)的关联，从而改变了通道门控。这些分子变化减少了MICU1/2依赖的MTCU的协同激活，从而减少了对Ca-m(2+)的摄取。此外，我们还表明，MCUB掺入MTCU是一种应激反应机制，可以限制心脏损伤期间的钙-m(2+)超载。事实上，MCUB的过度表达足以减少缺血/再灌注损伤后的心肌梗死面积。然而，MCUB掺入MTCU确实是有代价的；钙-m(2+)摄取的急剧下降损害了线粒体的能量和收缩功能。结论：我们详细描述了一种新的调节机制来调节MTCU功能和钙-m(2+)摄取。我们的结果表明，MTCU化学计量学中依赖于MCUB的变化是调节Ca-m(2+)摄取和细胞生理的重要调节机制。
Background: The mitochondrial calcium uniporter (mtCU) is an approximate to 700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (Ca-m(2+)) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. Methods: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. Results: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing Ca-m(2+) uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit Ca-m(2+) overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in Ca-m(2+) uptake impair mitochondrial energetics and contractile function. Conclusions: We detail a new regulatory mechanism to modulate mtCU function and Ca-m(2+) uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate Ca-m(2+) uptake and cellular physiology.