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Cysteines in the loop between IS5 and the pore helix of CaV3.1 are essential for channel gating

Authors
  • Karmazinova, Maria1
  • Beyl, Stanislav2
  • Stary-Weinzinger, Anna2
  • Suwattanasophon, Chonticha3
  • Klugbauer, Norbert4
  • Hering, Steffen2
  • Lacinova, Lubica1
  • 1 Slovak Academy of Sciences, Institute of Molecular Physiology and Genetics, Bratislava, Slovakia , Bratislava (Slovakia)
  • 2 University of Vienna, Institut für Pharmakologie und Toxikologie, Vienna, Austria , Vienna (Austria)
  • 3 University of Vienna, Institute of Theoretical Chemistry, Vienna, Austria , Vienna (Austria)
  • 4 Albert-Ludwigs Universität, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Freiburg, Germany , Freiburg (Germany)
Type
Published Article
Journal
Pflügers Archiv - European Journal of Physiology
Publisher
Springer-Verlag
Publication Date
Sep 09, 2010
Volume
460
Issue
6
Pages
1015–1028
Identifiers
DOI: 10.1007/s00424-010-0874-5
Source
Springer Nature
Keywords
License
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Abstract

The role of six cysteines of CaV3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in CaV3.1 gating. We hypothesize that cysteines in the large extracellular loop of CaV3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.

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