Contribution of Coiled-Coil Assembly to Ca 2+ /Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes
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 activi...
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Published in | Journal of the American Society of Nephrology Vol. 30; no. 9; p. 1587 |
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Main Authors | , , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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United States
01.09.2019
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Abstract | 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.
We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca
-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.
Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca
-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
elevations and a disorganized cytoskeleton.
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
may contribute to structural damage in the podocytes. |
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AbstractList | 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.
We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca
-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.
Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca
-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
elevations and a disorganized cytoskeleton.
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
may contribute to structural damage in the podocytes. |
Author | Sakaguchi, Reiko Polat, Onur K Tochio, Hidehito Reiser, Jochen Imamura, Kayo Uno, Masatoshi Mori, Masayuki X Itsuki, Kyohei Tran, Ha Nam Asanuma, Katsuhiko Mori, Yasuo Ariyoshi, Mariko Wong, Chee Fah Inoue, Ryuji Morii, Takashi Shirakawa, Masahiro Maruyama, Terukazu Ichikawa, Jun |
Author_xml | – sequence: 1 givenname: Onur K surname: Polat fullname: Polat, Onur K organization: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering – sequence: 2 givenname: Masatoshi orcidid: 0000-0001-5875-8445 surname: Uno fullname: Uno, Masatoshi organization: Department of Molecular Engineering, Graduate School of Engineering – sequence: 3 givenname: Terukazu surname: Maruyama fullname: Maruyama, Terukazu organization: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering – sequence: 4 givenname: Ha Nam surname: Tran fullname: Tran, Ha Nam organization: Department of Technology and Ecology, Laboratory of Environmental Systems Biology, Graduate School of Global Environmental Studies – sequence: 5 givenname: Kayo surname: Imamura fullname: Imamura, Kayo organization: Department of Biophysics, Graduate School of Science – sequence: 6 givenname: Chee Fah surname: Wong fullname: Wong, Chee Fah organization: Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, Perak, Malaysia – sequence: 7 givenname: Reiko surname: Sakaguchi fullname: Sakaguchi, Reiko organization: Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan – sequence: 8 givenname: Mariko surname: Ariyoshi fullname: Ariyoshi, Mariko organization: Department of Molecular Engineering, Graduate School of Engineering – sequence: 9 givenname: Kyohei surname: Itsuki fullname: Itsuki, Kyohei organization: Department of Physiology, Fukuoka University School of Medicine, Fukuoka, Japan – sequence: 10 givenname: Jun surname: Ichikawa fullname: Ichikawa, Jun organization: Department of Physiology, Fukuoka University School of Medicine, Fukuoka, Japan – sequence: 11 givenname: Takashi surname: Morii fullname: Morii, Takashi organization: Institute of Advanced Energy, Kyoto University, Kyoto, Japan – sequence: 12 givenname: Masahiro surname: Shirakawa fullname: Shirakawa, Masahiro organization: Department of Molecular Engineering, Graduate School of Engineering – sequence: 13 givenname: Ryuji surname: Inoue fullname: Inoue, Ryuji organization: Department of Physiology, Fukuoka University School of Medicine, Fukuoka, Japan – sequence: 14 givenname: Katsuhiko surname: Asanuma fullname: Asanuma, Katsuhiko organization: Department of Nephrology, School of Medicine, Chiba University, Chiba, Japan – sequence: 15 givenname: Jochen surname: Reiser fullname: Reiser, Jochen organization: Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois – sequence: 16 givenname: Hidehito surname: Tochio fullname: Tochio, Hidehito email: tochio@mb.biophys.kyoto-u.ac.jp organization: Department of Biophysics, Graduate School of Science, tochio@mb.biophys.kyoto-u.ac.jp – sequence: 17 givenname: Yasuo surname: Mori fullname: Mori, Yasuo organization: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering – sequence: 18 givenname: Masayuki X surname: Mori fullname: Mori, Masayuki X email: mxmori@med.uoeh-u.ac.jp organization: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, mxmori@med.uoeh-u.ac.jp |
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Keywords | chronic kidney disease electrophysiology podocyte Calcium signal focal segmental glomerulosclerosis TRPC channel |
Language | English |
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Snippet | TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude... |
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SubjectTerms | Actins - ultrastructure Animals Binding Sites Calcium - metabolism Calmodulin - genetics Calmodulin - metabolism Cytoskeleton - ultrastructure Gain of Function Mutation Glomerulosclerosis, Focal Segmental - genetics Glomerulosclerosis, Focal Segmental - metabolism HEK293 Cells Humans Mice Phenotype Podocytes Protein Domains TRPC6 Cation Channel - genetics TRPC6 Cation Channel - ultrastructure |
Title | Contribution of Coiled-Coil Assembly to Ca 2+ /Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes |
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