Charge movement and transcription regulation of L-type calcium channel α1S in skeletal muscle cells

Authors

  • Zhenlin Zheng,

    1. Departments of Physiology and Pharmacology,
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  • Zhong-Min Wang,

    1. Departments of Physiology and Pharmacology,
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  • Osvaldo Delbono

    Corresponding author
    1. Departments of Physiology and Pharmacology,
    2. Departments of Internal Medicine, Gerontology
    3. Departments of Neuroscience Program, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
    • Corresponding author Osvaldo Delbono: Department of Physiology and Pharmacology, Wake Forest Unkversity School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. Email: odelbono@wfubmc.edu

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Abstract

Several factors, such as Ca2+, trophic factors and ageing, regulate dihydropyridine-sensitive receptor (DHPR) α1 subunit expression. However, basic mechanisms of DHP α1S expression are unknown. To better understand the regulatory elements that control transcription, the 1.2 kb 5′-flanking region fragment immediately upstream of the mouse L-type Ca2+ channel or DHPR α1S gene was isolated and sequenced. Luciferase reporter constructs driven by different promoter regions of mouse DHPR α1S gene were used for transient transfection assays in muscle C2C12 cells. In these preparations we found that three regions corresponding to CREB, GATA-2 and SOX-5 consensus sequence within the 5′-flanking region of the DHPR α1S gene are important for DHPR α1S gene transcription. Antisense oligonucleotides against CREB, GATA-2 and SOX-5 significantly reduced charge movement in C2C12 cells. Charge movement was recorded in the whole-cell configuration of the patch clamp technique. Results from cells transfected with antisense (AS) and sense (S) oligonucleotides and nontransfected cells were compared. Charge movement experiments were fitted to a Boltzmann equation. Maximum charge movement (Qmax) (nC μF−1, mean ±s.e.m.) for S- and AS-CREB was 70.3 ± 2.9 and 52.8 ± 3.3, respectively (P < 0.05). The same parameter for S- and AS-GATA-2 was 71.3 ± 3.9 and 48.2 ± 2.3, respectively (P < 0.05) and for S- and AS-SOX-5 was 70.4 ± 4.2 and 45.1 ± 3.2, respectively (P < 0.05). Values recorded in cells transfected with sense S-CREB, S-GATA-2 and S-SOX-5 oligonucleotides were not significantly different from those recorded in nontransfected cells. This study demonstrates that the transcription factors CREB, GATA-2 and SOX-5 play a significant role in the expression of the skeletal muscle DHPR or L-type Ca2+ channel α1S.

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