Transcriptional regulation of α1H T-type calcium channel under hypoxia

Hassan Sellak, Chun Zhou, Bainan Liu, Hairu Chen, Thomas M. Lincoln, Songwei Wu

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

The low-voltage- activated T-type Ca2+ channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site 1,173CACGC 1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.

Original languageEnglish (US)
Pages (from-to)C648-C656
JournalAmerican Journal of Physiology - Cell Physiology
Volume307
Issue number7
DOIs
Publication statusPublished - Oct 1 2014

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Keywords

  • Gene expression
  • Hypoxia
  • Hypoxia-response element
  • T-type calcium channel

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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