PC2 (polycystin-2) forms a Ca2+-permeable channel in the cell membrane and its function is regulated by cytosolic Ca2+ levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca2+-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca2+ regulates the channel through the HPC2 Cterm, we characterized Ca2+ binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen–deuterium (H–D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca2+. The HPC2 L1EF construct contains the Ca2+-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca2+-bound conformation of the EF-hand, thus enhancing its Ca2+-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca2+, we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca2+ binding. Nevertheless, they have different profiles of H–D exchange under non-saturating Ca2+ conditions, implying their different conformational exchange between the Ca2+-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca2+-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2’s roles in the pathophysiology of polycystic kidney disease.
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Point scanning confocal microscopic imaging of C2C12 undifferentiatedmyoblasts (top panels) and differentiatedmultinucleated myotubes (bottom panels) immunofluorescently labelled with DAPI (blue) PDLIM7 (green) and Phalloidin or Nedd4-1 (red). Merged images show co-localization of PDLIM7 and Phalloidin decreases with myotube formation (left panels) and co-location of PDLIM7 and Nedd4-1 increases with myotube formation (right panels). Image courtesy of Robert D’Cruz et al. (for further details see pages 267–276). - PDF Icon PDF LinkFront Matter
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Research Article|
January 25 2016
Conformational dynamics of Ca2+-dependent responses in the polycystin-2 C-terminal tail
Yifei Yang;
Yifei Yang
*Department of Laboratory Medicine, Yale University, New Haven, CT 06520, U.S.A.
†Department of Pharmacology, Yale University, New Haven, CT 06520, U.S.A.
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Michael E. Hodsdon;
Michael E. Hodsdon
*Department of Laboratory Medicine, Yale University, New Haven, CT 06520, U.S.A.
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Elias J. Lolis;
Elias J. Lolis
1
†Department of Pharmacology, Yale University, New Haven, CT 06520, U.S.A.
1Correspondence may be addressed to either of these authors (email elias.lolis@yale.edu or barbara.ehrlich@yale.edu).
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Barbara E. Ehrlich
Barbara E. Ehrlich
1
†Department of Pharmacology, Yale University, New Haven, CT 06520, U.S.A.
‡Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06520, U.S.A.
1Correspondence may be addressed to either of these authors (email elias.lolis@yale.edu or barbara.ehrlich@yale.edu).
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Publisher: Portland Press Ltd
Received:
September 30 2015
Revision Received:
November 13 2015
Accepted:
November 16 2015
Accepted Manuscript online:
November 16 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (3): 285–296.
Article history
Received:
September 30 2015
Revision Received:
November 13 2015
Accepted:
November 16 2015
Accepted Manuscript online:
November 16 2015
Citation
Yifei Yang, Michael E. Hodsdon, Elias J. Lolis, Barbara E. Ehrlich; Conformational dynamics of Ca2+-dependent responses in the polycystin-2 C-terminal tail. Biochem J 1 February 2016; 473 (3): 285–296. doi: https://doi.org/10.1042/BJ20151031
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