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Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells

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Abstract

Large-conductance Ca2+-activated K+ channel is formed by a tetramer of the pore-forming α-subunit and distinct accessory β-subunits (β1–β4) which contribute to BKCa channel molecular diversity. Accumulative evidences indicate that not only α-subunit alone but also the α + β subunit complex and/or β-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of α + β1 subunit complex or β1-subunit in BKCa channel, we established an expression system that reliably coexpress hSloα + β1 subunit complex in HEK293 cells. The coexpression of hSloα + β1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloα + β1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloα + β1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca2+-sensitivity, voltage-dependency, high conductance (151 ± 7 pS), and its pharmacological activation and inhibition.

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Acknowledgements

We thank Dr. J.D. Lippiat (Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, UK) for a gift of the pIRES-hSloα + β1 construct. We also thank Dr. Tao Wang (Department of Immunology, Fourth Military Medical University, China) for advice and providing HEK293 cell line. This study was supported by National Science Found of China (No. 30600215) and Military Health Ministry of China (No. 06Q058).

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Authors and Affiliations

  1. Department of Aerospace Physiology, Fourth Military Medical University, 17#, Changle West Road, Xi’an, 710032, China

    Jun Li, Chang-Lei Deng, Fang Gao, Jiu-Hua Cheng, Zhi-Bin Yu & Man-Jiang Xie

  2. Department of Aerospace Clinical Medicine, Fourth Military Medical University, 17#, Changle West Road, Xi’an, 710032, China

    Jun Li & Li Liu

  3. Key Laboratory of Aerospace Medicine, Ministry of China, Xi’an, 710032, China

    Jun Li, Chang-Lei Deng, Fang Gao, Jiu-Hua Cheng, Zhi-Bin Yu, Li Liu & Man-Jiang Xie

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  1. Jun Li
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  2. Chang-Lei Deng
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  4. Jiu-Hua Cheng
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Correspondence to Li Liu or Man-Jiang Xie.

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Jun Li and Chang-Lei Deng contributed equally to this study.

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Li, J., Deng, CL., Gao, F. et al. Coexpression and characterization of the human large-conductance Ca2+-activated K+ channel α + β1 subunits in HEK293 cells. Mol Cell Biochem 331, 117–126 (2009). https://doi.org/10.1007/s11010-009-0149-7

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