OBJECTIVE: Despite the primacy of the sodium current in cardiac electrophysiology and evidence that decreased sodium current is arrhythmogenic in humans, little is known about transcriptional regulation of the underlying gene, SCN5A. METHODS: We have cloned a 2.7 kb segment of 5'-flanking region of SCN5A and identified multiple transcription initiation sites by primer extension and RNase protection. Transient transfection assays in neonatal mouse myocytes and in Chinese Hamster Ovary (CHO) cells were employed to identify promoter activities. PCR-single stranded conformational polymorphism (SSCP) analysis was used to screen DNA variants in the promoter region. RESULTS: The fragment includes >2 kb of upstream sequence, the 173-bp non-coding exon 1, and a portion of the 16-kb intron 1; the region is highly GC-rich and TATA-less. Transient transfection assays in neonatal mouse myocytes and in CHO cells identified (1) a core promoter in the -261/+140 segment, (2) regions conferring approximately 3-fold decreases from core promoter activity in the 5' upstream region (-261/-454 and -1020/-2109), and approximately 3-fold increases in intron 1 (+255/+410 and+539/+613), and (3) a very strong negative regulatory region between +613 and +754 in intron 1. A core promoter polymorphism, present in 6/142 (4%) of normal alleles screened, increased reporter activity approximately 50% in myocytes but not in CHO cells. CONCLUSION: The SCN5A promoter includes multiple positive and negative cis-acting elements extending into intron 1. A common polymorphism in this region modulates channel expression in vitro.
OBJECTIVE: Despite the primacy of the sodium current in cardiac electrophysiology and evidence that decreased sodium current is arrhythmogenic in humans, little is known about transcriptional regulation of the underlying gene, SCN5A. METHODS: We have cloned a 2.7 kb segment of 5'-flanking region of SCN5A and identified multiple transcription initiation sites by primer extension and RNase protection. Transient transfection assays in neonatal mouse myocytes and in Chinese Hamster Ovary (CHO) cells were employed to identify promoter activities. PCR-single stranded conformational polymorphism (SSCP) analysis was used to screen DNA variants in the promoter region. RESULTS: The fragment includes >2 kb of upstream sequence, the 173-bp non-coding exon 1, and a portion of the 16-kb intron 1; the region is highly GC-rich and TATA-less. Transient transfection assays in neonatal mouse myocytes and in CHO cells identified (1) a core promoter in the -261/+140 segment, (2) regions conferring approximately 3-fold decreases from core promoter activity in the 5' upstream region (-261/-454 and -1020/-2109), and approximately 3-fold increases in intron 1 (+255/+410 and+539/+613), and (3) a very strong negative regulatory region between +613 and +754 in intron 1. A core promoter polymorphism, present in 6/142 (4%) of normal alleles screened, increased reporter activity approximately 50% in myocytes but not in CHO cells. CONCLUSION: The SCN5A promoter includes multiple positive and negative cis-acting elements extending into intron 1. A common polymorphism in this region modulates channel expression in vitro.
Authors: K M Giacomini; C M Brett; R B Altman; N L Benowitz; M E Dolan; D A Flockhart; J A Johnson; D F Hayes; T Klein; R M Krauss; D L Kroetz; H L McLeod; A T Nguyen; M J Ratain; M V Relling; V Reus; D M Roden; C A Schaefer; A R Shuldiner; T Skaar; K Tantisira; R F Tyndale; L Wang; R M Weinshilboum; S T Weiss; I Zineh Journal: Clin Pharmacol Ther Date: 2007-03 Impact factor: 6.875
Authors: Thomas C Atack; Dina Myers Stroud; Hiroshi Watanabe; Tao Yang; Lynn Hall; Susan B Hipkens; John S Lowe; Brenda Leake; Mark A Magnuson; Ping Yang; Dan M Roden Journal: Circ Res Date: 2011-05-12 Impact factor: 17.367
Authors: Elizabeth A Schroder; Mellani Lefta; Xiping Zhang; Daniel C Bartos; Han-Zhong Feng; Yihua Zhao; Abhijit Patwardhan; Jian-Ping Jin; Karyn A Esser; Brian P Delisle Journal: Am J Physiol Cell Physiol Date: 2013-01-30 Impact factor: 4.249