Transcriptional regulation of α_1H T-type calcium channel under hypoxia

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, α_1G, α_1H, and α_1I, only α_1H 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 α_1G and α_1H 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 α_1H mRNA at 12 h along with a significant rise in α_1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α_1G and α_1H 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 α_1H, but not α_1G, 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 α_1H 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 α_1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α_1H promoter region.