Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes

Significance Statement TRPC6 is a receptor-activated nonselective cation channel. Naturally occurring mutations in this channel are associated with FSGS. FSGS-associated TRPC6 channel mutations appear to have a “gain-of-function” mechanism, but the exact mechanism remains unclear. The authors show t...

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Published in:Journal of the American Society of Nephrology 2019-09, Vol.30 (9), p.1587-1603
Main Authors: Polat, Onur K., Uno, Masatoshi, Maruyama, Terukazu, Tran, Ha Nam, Imamura, Kayo, Wong, Chee Fah, Sakaguchi, Reiko, Ariyoshi, Mariko, Itsuki, Kyohei, Ichikawa, Jun, Morii, Takashi, Shirakawa, Masahiro, Inoue, Ryuji, Asanuma, Katsuhiko, Reiser, Jochen, Tochio, Hidehito, Mori, Yasuo, Mori, Masayuki X.
Format: Article
Language:English
Subjects:
Basic Research
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Summary:Significance Statement TRPC6 is a receptor-activated nonselective cation channel. Naturally occurring mutations in this channel are associated with FSGS. FSGS-associated TRPC6 channel mutations appear to have a “gain-of-function” mechanism, but the exact mechanism remains unclear. The authors show that negative feedback regulation induced by cellular calcium levels, called Ca 2+ -dependent inactivation (CDI), is impaired in FSGS-associated TRPC6 channel mutations. Dysfunctional coiled-coil assembly in the mutated TRPC6 disrupts calmodulin bridging which is essential for CDI. The authors reveal how disruption in a Ca 2+ -dependent regulatory mechanism may play a role in FSGS. 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 Ca 2+ -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 Ca 2+ -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 Ca 2+ 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 Ca 2+ may contribute to structural damage in the podocytes.
ISSN:1046-6673
1533-3450
DOI:10.1681/ASN.2018070756