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

  • Ion channels, receptors and transporters
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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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Acknowledgment

Supported by grants from VEGA 2/0195, VVCE-0064-07, Center of Excellence for Cardiovascular Research SAS (LL) and the DFG (NK). The authors thank Emilia Kocurova and Ute Christoph for skillful technical assistance and Martina Kurejova for help with construction of C241A and C298A mutants and Eugen Timin for helpful comments on the manuscript.

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No specific ethical issues are related to reported experiments.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia

    Maria Karmazinova & Lubica Lacinova

  2. Institut für Pharmakologie und Toxikologie, University of Vienna, Vienna, Austria

    Stanislav Beyl, Anna Stary-Weinzinger & Steffen Hering

  3. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs Universität, Freiburg, Germany

    Norbert Klugbauer

  4. Institute of Theoretical Chemistry, University of Vienna, Vienna, Austria

    Chonticha Suwattanasophon

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  1. Maria Karmazinova
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  2. Stanislav Beyl
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  4. Chonticha Suwattanasophon
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  5. Norbert Klugbauer
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Correspondence to Lubica Lacinova.

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Karmazinova, M., Beyl, S., Stary-Weinzinger, A. et al. Cysteines in the loop between IS5 and the pore helix of CaV3.1 are essential for channel gating. Pflugers Arch - Eur J Physiol 460, 1015–1028 (2010). https://doi.org/10.1007/s00424-010-0874-5

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