title: Informatic and functional approaches to identifying a regulatory region for the cardiac sodium channel.
abstract: BACKGROUND
  Although multiple lines of evidence suggest that variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation.
  
  
  OBJECTIVE
  We used in silico and in vitro experiments to identify possible noncoding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted.
  
  
  RESULTS
  We identified 92 noncoding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved noncoding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as a potential regulatory region. Using recombinase-mediated cassette exchange (RMCE), we generated mice in which a 435-base pair region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, Na(V)1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2 ms in controls versus 9.7±0.2 ms in knockouts), indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3' segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6%±0.3% of baseline in CHO cells and 16%±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62%±4% and 57%±2% of baseline in CHO cells and myocytes, respectively (both P<0.05).
  
  
  CONCLUSIONS
  These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a noncoding region underlying this regulation.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Atack
authors.forename: Dina Myers
authors.initials: DM
authors.lastname: Stroud
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.forename: Lynn
authors.initials: L
authors.lastname: Hall
authors.forename: Susan B
authors.initials: SB
authors.lastname: Hipkens
authors.forename: John S
authors.initials: JS
authors.lastname: Lowe
authors.forename: Brenda
authors.initials: B
authors.lastname: Leake
authors.forename: Mark A
authors.initials: MA
authors.lastname: Magnuson
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.forename: Dan M
authors.initials: DM
authors.lastname: Roden
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D004562:Electrocardiography; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels