Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes
Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes
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DOI：
10.1681/asn.2018070756
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发表时间：
2019-09-01
影响因子：
13.6
通讯作者：
Mori, Masayuki X.
中科院分区：
文献类型：
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作者：
Polat, Onur K.;Uno, Masatoshi;Mori, Masayuki X.
TRPC6是一种非选择性阳离子通道，该基因的突变与FSGS有关。这些突变与TRPC6电流振幅放大和/或通道失活延迟（功能获得表型）有关。然而，TRPC6活性的功能获得机制尚未明确。方法通过电生理、生化和生物物理实验来阐明钙调素（CaM）介导的TRPC6 Ca2+依赖性失活（CDI）的分子机制。为了解决CDI的病理生理贡献，我们评估了培养小鼠足细胞中肌动蛋白丝的组织。结果CaM双叶均对CDI有促进作用。此外，CaM与TRPC6 CaM结合域（CBD）的结合是Ca2+依赖的，并表现出1:2 （CaM/CBD）的化学计量。TRPC6卷曲线圈组件使两个cbd足够接近，对于CDI至关重要。去除螺旋状结构会减缓TRPC6的CDI，这表明螺旋状结构在两个CBDs上配置CaM结合的两个叶来诱导正常的CDI。线圈内与fsgs相关的TRPC6突变严重延迟了CDI，并经常增加TRPC6电流幅度。在培养的小鼠足细胞中，fsgs相关通道和CaM突变导致持续的Ca2+升高和细胞骨架紊乱。结论在fsgs引起的TRPC6突变中发现的功能获得机制可以通过CDI的损伤来解释，CDI是由TRPC的卷曲线圈组装被破坏引起的，而这是CaM结合所必需的。由此产生的过量Ca2+可能导致足细胞的结构损伤。
Background TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.Methods We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.Results Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.Conclusions The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.