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Cardiovascular Research 2004 61(1):56-65; doi:10.1016/j.cardiores.2003.09.030
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Copyright © 2004, European Society of Cardiology

Cloning and initial characterization of the human cardiac sodium channel (SCN5A) promoter *

Ping Yanga, Sabina Kupershmidta,b and Dan M. Roden*,a,c

aDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
bDepartment of Anesthesiology, Vanderbilt University School of Medicine, USA
cDepartment of Medicine, Vanderbilt University School of Medicine, USA

* Corresponding author. Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University School of Medicine, 532 Medical Research Building I, Nashville, TN 37232, USA. Tel.: +1-615-322-0067; fax: +1-615-343-4522. dan.roden{at}vanderbilt.edu

Received 18 June 2003; revised 17 September 2003; accepted 23 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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 ~3-fold decreases from core promoter activity in the 5' upstream region (–261/–454 and –1020/–2109), and ~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 ~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.

KEYWORDS Ion channels; Arrhythmia (mechanism); Gene polymorphisms


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ion channel diseases ("channelopathies") cause multiple disease phenotypes, including cardiac arrhythmias [1–3], myotonias [4,5], epilepsies [4–7], hypoglycemia [8,9], electrolyte abnormalities [10], and hypertension [11,12]. Disease-associated mutations disrupt channel function through multiple mechanisms, including synthesis of truncated channels, misprocessing, and altered gating and selectivity. Voltage-gated sodium channels are the pore-forming transmembrane proteins that produce the large inward current responsible for the rapid upstroke of the action potential in many tissues. These channels are encoded by a genetic superfamily that includes 11 members encoding distinct sodium channels; SCN5A (Nav1.5) [13] encodes the cardiac-specific isoform. Mutations in SCN5A are one cause of the congenital long QT syndrome; these mutations alter channel gating to increase inward plateau current, thereby prolonging QT [14]. In addition, multiple lines of evidence indicate that loss of sodium channel function is also highly arrhythmogenic. For example, chronic therapy with sodium channel blocking drugs in patients convalescing from myocardial infarction increased total mortality and sudden cardiac death (SCD) likely due to arrhythmias [15,16]. Mutations in SCN5A that generate truncated, misprocessed, or dysfunctional proteins produce the Brugada variant of idiopathic ventricular fibrillation [17]; one interesting aspect of this entity is that patients may remain asymptomatic until challenged with a sodium channel blocking agent, when the clinical phenotype becomes more apparent. These data, taken together, suggested to us the hypothesis that SCN5A expression might vary among individuals but that this variability might remain inapparent until the superimposition of stressors such as sodium channel blockers or myocardial ischemia. As a first step in testing this concept, we have now cloned the promoter region of human SCN5A, mapped and characterized cardiac cis-acting DNA regulatory elements in vitro, and identified a polymorphism with in vitro functional consequences in the core promoter.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Initial database screening and cloning
The translation start site for human SCN5A is in exon 2, and previous studies have identified promoter elements in the rat tetrodotoxin-resistant skeletal muscle sodium channel (SkM2), an ortholog to SCN5A [18,19]. Therefore, our initial approach was to search for human SCN5A genomic flanking sequences assuming homology to rSkM2 (Genbank, L11243 [GenBank] ). However, neither the public nor the Celera databases include human SCN5A flanking sequence. A BLAST search [20] using the rat SkM2 promoter against the Celera human genome database yielded three short matches on the GA_x8W4M44 scaffold in the Celera database (Fig. 1). Primer set 1F/1R was used to amplify the gap in the sequence (all primers used in this study are listed in Table 1). The mouse SCN5A promoter region and exon 1 are similarly not included in any available databases, so we cloned ~2 kb of 5' flanking region of mouse SCN5A using the same strategy, using primer pairs m1F/m1R. PCR reactions unless otherwise specified were carried out by using the LA PCR Kit (Ver2.1 TaKaRa) according to the manufacturer's instructions. PCR reactions were performed at 95 °C for 1 min followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min, and a final 7 min extension at 72 °C. The sequences of all PCR products and vector inserts in this study were verified using dye-terminator chemistry.


Figure 1
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  		Fig. 1 Strategy for identification of 5'-flanking sequence in the human cardiac sodium channel gene. After determining that the sequence was absent from both the public and the Celera databases, the previously reported rSkM2 sequence (A; GenBank accession number: L11243) was used in a BLAST search of the Celera human database. This search yielded three short matches ("hits"), indicated by the open boxes, on Celera scaffold GA_x8W4M44 (B). Further PCR generated the –2187/+613 fragment, a starting point for the present studies. The bottom diagram (C) shows the locations of the primer used for primer extension and the RNase protection probe used to define the transcription initiation sites, and thus the 5' end of exon 1 (see Fig. 2).

 

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  		Table 1 PCR primer set

 
2.2 Mapping transcription initiation sites
Two approaches, primer extension and RNase protection, were used. 5' Primer extension analysis was carried out using the Primer Extension System (Promega). Briefly, poly(A) mRNA was isolated from human heart using mRNA purification kits (Poly(A) Pure Kit, Ambion). Primer extension was performed using a 5'-end [{gamma}-32P] labeled gene-specific primer designed to be complementary to the human SCN5A mRNA sequence from –8 to –31 relative to the first nucleotide of the previously reported exon 1 (5'-AGCGATCCCTGCATCCTACGGGCG-3') [21]. The resulting extension products were resolved on a 7 M urea/8% polyacrylamide gel alongside 1-bp DNA size markers.

RNase protection analysis was carried out as previously described [22]. A genomic DNA fragment complementary to the –85 to +118 region, relative to the major transcription start site determined by primer extension above, was amplified. PCR products were purified through a QIAGEN column and subcloned into the pGEM-T Easy Vector (Promega). This template DNA was linearized with SpeI and an antisense cRNA probe was subsequently synthesized by using the SP6 promoter. The radiolabeled probe (~5 x 105 cpm/ml) was gel purified and hybridized with 5 µg of the human heart mRNA or the same amount of yeast tRNA controls in 80% formamide, 400 mM NaCl, and 40 mM PIPES (pH 6.4). The hybridization reaction was heated to 85 °C for 10 min then slowly cooled to 45 °C overnight. Samples were then digested with 7.5 µg RNase A at 30 °C for 45 min. The protected fragments were separated on a 6% polyacrylamide gel with a DNA size ladder and imaged by a 48-h exposure of X-ray film at –70 °C.

As described below, multiple transcription initiation sites were identified in SCN5A. In this report, position +1 is designated the major transcription initiation site determined by RNase protection. MatInspector v.2.2 (http://www.genomatix.de/software_services/software/MatInspector/matinspector.html) was used to search the sequence for consensus transcription factor binding sites.

2.3 Transient transfection assays
Human genomic DNA was used as a template to amplify two fragments which contained the –2190 to +613 segment (designated as P1) and the –451 to +745 segment (designated as P2), using primer sets P1F/P1R and P2F/P2R. Two series of constructs, one extending 5' from position +140 and the other 3' from position –261, were generated for subsequent deletion analysis. For deletion analysis using constructs anchored at +140 and of variable 5' lengths, the P1 fragment was cloned into the pGEM-T Easy vector. The plasmid was then digested with NcoI and SacI, and the insert was subcloned into the pGL3-Basic vector (Promega), which contains the luciferase coding sequence to generate a SCN5A promoter-luciferase fusion construct designated as pGL-P1. pGL-P1 was then used as a template to generate a series of promoter constructs with varying upstream boundaries generated by PCR, using forward primers containing the SacI recognition sequence at the 5' end and reverse primer containing the NcoI recognition sequence (underlined; Table 1). PCR products were digested with SacI and NcoI, and the fragments were subcloned into SacI/NcoI-digested pGL3-Basic vector. The same strategy was used to derive a series of constructs from pGL-P2 for 3' orientation deletion analysis.

Reporter activity was assayed in neonatal mouse myocytes and in CHO cells. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). One-day-old B6D2 mice were sacrificed by dousing in ethanol and decapitation. Neonatal hearts were removed and placed in 1 x PBS solution. Ventricular segments were digested by Trypsin-Versene (Biofluids), and cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% (v/v) NuSerum (Beckton Dickinson), 2.5 mM thymidine, and penicillin–streptomycin (10 units/ml and 10 mg/ml, respectively) in a humidified 5% CO2 atmosphere at 37 °C. Cells were allowed to attach for 48 h before being used. Chinese hamster ovary K1 (CHO-K1) cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as previously described [23].

The SCN5A promoter/luciferase fusion genes (1 µg DNA) were transfected into the neonatal mouse cardiac cells using Fugene 6 (Roche), and into CHO cells using lipofectamine reagent (Invitrogen). In each experiment, the pRL-TK plasmid (0.05 µg), encoding Renilla luciferase (Promega) was co-transfected to normalize for experimental variability caused by differences in cell viability or transfection efficiency. Luminescence was measured after 48-h transfection by using Dual-Luciferase Reporter Assay System (Promega). The pGL3-Basic (promoterless) plasmid was tested in each experiment and its activity level served as the baseline.

2.4 Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from primary myocytes and from CHO cells were prepared as previously reported [29]. Double-stranded oligonucleotide-containing potential cis-acting elements were annealed, end-labeled with 32P-dATP, and incubated with nuclear extracts. EMSA reactions were established in 20 µl for 30 min at room temperature, using 5 µg nuclear extract and 8 fmol end-labeled oligonucleotide double-stranded cassette in 10% (v/v) glycerol and 1 µg poly (dI:dC). The reaction buffer (pH 7.9) contained 25 mM HEPES, 100 mM KCl, and 25 mM DTT. Complexes were resolved using a 6% non-denaturing polyacrylmide gel in 1 x TBE buffer and electrophoresis at 10 V/cm for 3–5 h. Gels were dried and imaged by a 48-h exposure of X-ray film at –70 °C.

2.5 Polymorphism screening
The core promoter described below was screened for DNA variants in 71 anonymized human samples of mixed ethnicity. The investigation conforms with the principles outlined in the Declaration of Helsinki. An approximately 2.8-kb fragment flanking the transcriptional start sites was first amplified with primer pair 1F/1R. The PCR product was diluted 1/50 and used as template for a set of subsequent nested PCR reactions that generated PCR products <350 bp for single-stranded conformational polymorphism (SSCP) analysis (primer sets, see Table 1). SSCP was performed on 0.5 x mutation detection enhancement gels (BioWhittaker Molecular Applications) that were electrophoresed overnight at 3 W and stained with silver nitrate. Abnormal SSCP conformers were excised from the dried gels and eluted into sterile water. Eluted DNA was re-amplified using original primers, purified by spin column chromatography (Qiagen), and sequenced. Mutant constructs were generated by site-directed mutagenesis using the QuickChange (Stratagene) kit.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Mapping of transcription initiation sites
Both primer extension and RNase protection identified multiple transcription initiation sites clustered within a 50-nucleotide region (Fig. 2), homologous to that identified in rat (Fig. 3). Some sites identified by the two methods overlapped (Fig. 2). The major transcription start site identified by RNase protection was 4 bp longer than the most 5' one identified by primer extension, and the major transcription start site identified by RNase protection was designated as +1. Identifying these transcription initiation start sites allowed us to determine that the non-coding human SCN5A exon 1 is 173 bp, 76 bp longer than previously reported [21]. Based on these data and the Celera scaffold, intron 1 is ~16 kb. Thus both the parental pGL-P1 and pGL-P2 constructs used for reporter experiments included 5'-flanking sequence, exon 1, and a portion of intron 1.


Figure 2
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  		Fig. 2 Mapping of transcription initiation sites. (A) Ribonuclease protection analysis. Lane 1: markers; Lane 2: 5 µg human heart mRNA. Lane 3: 10 µg yeast tRNA control. (B) Analysis of primer extension products. Lane 1: marker, Lane 2: extension products. The solid arrow indicates the major transcription initiation site identified by RNase protection (A). Dotted arrows indicate transcription initiation sites identified both by RNase protection (A) and primer extension (B). (C) Nucleotide sequence of the transcription start sites. Stars (*) indicate transcription start sites identified by both methods and the number +1 indicates the major transcription initiation site identified by RNase protection (panel A).

 

Figure 3
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  		Fig. 3 Sequence alignments of human, rat, and mouse sequences. The 5' flanking sequences are highly conserved. The major transcription initiation site of human SCN5A identified by RNase protection is indicated by {blacktriangledown}; the major transcription initiation site identified by primer extension is indicated by {bigtriangledown}; and major transcription initiation site reported for rat rSkM2 is indicated by {diamondsuit}. The putative SP1 binding sites (GC boxes) and MyoD-binding site (E box) are shown. The gray box shows the site of the C-rich motif GACCCCGCCCC (–39 to –49) that consists of an overlapping CACC-like box and consensus GC box was absent in the mouse and rat sequences. C-92 is indicated by bullet.

 
3.2 Identification of the human SCN5A basal and tissue-specific promoters
Fig. 4 shows the results of promoter–reporter experiments using constructs of variable length 5' to position +140 in exon 1. The P+47 (i.e. +47 to +140) construct expressed near-background (<2-fold change vs. background) luciferase activity while activity of P+12 construct was increased 4.9–6.7-fold over background, identifying a basal promoter in this region. Activity of the P-261 construct was 21.4±3.2-fold increased over background in neonatal mouse myocytes vs. an 8.2±2.0-fold increase in CHO cells; this result suggests cardiac tissue-specific regulatory elements in this proximal 5'-flanking region. Constructs larger than –261 actually decreased reporter activity, suggesting negative regulatory elements further upstream, notably in the –454 to –261 and the –2109 to –1020 segments.


Figure 4
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  		Fig. 4 Analysis of human SCN5A promoter activities in CHO cells and neonatal mouse cardiac myocytes. The 5' deletion constructs used are depicted schematically on the left. Firefly luciferase expression levels, which report the activities of the inserted SCN5A sequence were divided by co-expressed Renilla luciferase activities and expressed as relative luciferase units. Data are presented and mean±S.E. (vs. empty vector), based on at least five separate transient transfection experiments.

 
This promoter region does not include a TATA box and is very highly GC-rich (>80% from –262 to +254). In the first 110 bp immediately 5' from the major transcription initiation site, there are four GC-boxes that resemble the consensus Sp1 binding site (GGGCGG) [19], at –29/–34, –40/–45, –51/–56, and –101/–106; four overlapping CACC-like boxes between –15 and –52 bp; and an E box, a MyoD-binding site [24], at –131 bp. These cis-regulatory elements are structurally homologous among human, mouse and rat (Fig. 3). These sequence identities support the idea that the Sp1 sites, previously identified as key regulators of expression of rSkM2 [18], play a role in expression of human SCN5A. One difference between the human and the rodent sequences is the presence of an extra 11 base pair C-rich motif GACCCCGCCCC in human (Fig. 3). When this motif was mutated to GACTCCGTCCC, wild-type promoter activity was reduced by 65% in neonatal mouse myocyte, and by 47% in CHO cells (Fig. 6A).


Figure 6
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  		Fig. 6 (A) Analysis of a C-rich motif in the core promoter of SCN5A (gray box in Fig. 3). The wild type and mutant constructs were transiently transfected and the data presented as in Fig. 4 (n = 5). (B) Functional analysis of a putative GATA-1 box at position +542/+545. The sequence was mutated to TTGC, and transcriptional activity assayed in the –261/+613 background. Data are presented as in Fig. 4 (n = 5–8). (C) Functional analysis of the C-92A polymorphism. Transcriptional activity was assayed on the –261/+255 background. Data are presented as in Fig. 4 (n = 8–10).

 
3.3 Regulatory sequences in intron 1
Previous studies with rSkM2 used cDNA as a starting point to clone 5' regulatory sequences. The cloning of genomic sequence allowed us to investigate potential cis-acting regulatory elements in intronic sequence. Fig. 5 shows the results of analyzing constructs of variable length 3' to position –261. The segment –261/+410 increased luciferase activity 2.0-fold over the core promoter (–261 to +140), and 3' extension of this sequence to +613 further increased luciferase expression, to 2.7-fold over the core promoter. Thus, these intronic sequences include positive elements located within segments +255/+410 and +539/+613. The –261/+613 construct increased expression 54.1±12.5-fold (n = 10) over background in myocytes; however, 3' extension to generate the slightly larger –261/+754 construct totally suppressed expression activity (3.2±0.8-fold). Thus, the gene includes a very strong negative regulatory element located within the +613 and +754 region. As shown in Fig. 5, these changes were near-absent in CHO cells. A potential GATA-1 factor binding site (5'-(A/T)GATA(A/G)-3') is located at position +542/+545 relative to the major transcription start site. Mutation of the wild-type motif GATA to TTGC in the SCN5A promoter/luciferase fusion gene containing the –261/+613 segment resulted in a 45% decrease (back to the core promoter level) in myocytes, with little change in the basal level of expression seen in CHO cells (Fig. 6B).


Figure 5
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  		Fig. 5 Analysis of promoter activity of SCN5A constructs 3' to the transcription initiation site. Data analysis and presentation as in Fig. 4.

 
Electrophoretic mobility shift assays were performed using (1) the evolutionarily conserved –54/–26 region containing two copies of the C-rich motif discussed above; (2) the predicted positive regulatory element within intron 1 from +540 to +613; and (3) the predicted negative element within intron 1 from +614 to +754. As shown in Fig. 7, each experiment identified bands present in experiments with neonatal myocytes or CHO cells, as well as myocyte-specific bands.


Figure 7
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  		Fig. 7 Electrophoretic mobility shift assays of potential DNA–protein interactions. Nuclear extracts were prepared from both cardiac myocytes and non-cardiac (CHO) cells. Nuclear extracts were tested with three predicted cis-acting elements identified in Figs. 4 and 5Go. DNA only lanes (not shown) displayed only rapidly migrating bands, as indicated by dotted arrows at the bottom of the figures. The solid arrows indicate shifted bands present only with the cellular extracts: the heavy arrows identify bands present only in myocytes, while the lighter ones show bands detected in both myocytes and CHO cells. (A) The evolutionarily conserved –54/–26 region containing two copies of the C-rich motif. (B) The predicted positive regulatory element within intron 1 from +540 to +613. (C) The predicted negative element within intron 1 from +614 to +754.

 
3.4 A DNA variant in the core promoter
SSCP analysis identified a C to A transversion at position –92 in 6/142 (4.2%) alleles; no homozygotes were identified. This polymorphism is located in the core promoter in a region highly conserved among human, rat, and mouse (Fig. 3). This change results in elimination of a consensus AP2 binding site (CCCG) and generation of a novel nuclear factor 1 consensus binding site (TGGC). A basal promoter construct (–261/+255) containing the minor A allele increased luciferase activity in neonatal cardiac myocytes from 21.8±2.5- to 31.1±3.3-fold (+43%, n = 9, P<0.05), but not in CHO cells (4.3±0.7- vs. 4.0±0.5-fold, n = 8, Fig. 6C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Sheng et al. [19] analyzed transcriptional control of the rat skeletal muscle subtype 2 (rSkM2), an ortholog of hSCN5A. We have taken advantage of their findings, and of conservation of pathways regulating gene expression across species, to clone the homologous regions in human and mouse, and to identify human SCN5A regulatory elements. Further, we identified multiple cis-acting elements—including previously unidentified intronic sequences—that regulate expression of SCN5A in a primary cardiac myocyte culture system. As is often the case with basal promoters that lack TATA boxes, multiple transcription initiation sites were identified within a short region.

Our combined 5' and 3' deletion analyses indicate that the core promoter is located between nucleotide sequence –261 and +140. This conserved regulatory element is highly GC-rich, characteristic of housekeeping genes. As in rat scn5a, the segment immediately upstream of the major transcription start site contains three GC boxes that could serve as binding sites for the Sp1 transcriptional factor, four identical C-rich motifs ({C/G}A{C/G}C) which are homologous to the CACC boxes that are often important for muscle restricted genes [25–28], and an E-box binding site for basic helix-loop-helix (bHLH) transcription factors of the muscle-specific MyoD family. The human sequence also includes an additional C-rich motif that we identified here as a regulator of expression in myocytes. Interestingly, a recent study identified this motif in the human cardiac troponin I promoter and found it is a binding site for as-yet-unidentified cardiac-restricted transcription factors [29]. Further, we have implicated a GATA family binding site in intron 1 as a positive regulatory region. The GATA family of transcription factors share a highly conserved DNA binding domain of two zinc fingers, which bind preferentially to the nucleotide sequence element 5'-(A/T)GATA(A/G)-3'. GATA-4, -5, and -6 have been implicated as key regulators of gene expression in the heart and gut. Our results also indicate other key regulatory sequences notably a strong negative element in the +613/+754 segment.

The results of this study represent a key first step in studies of regulation of expression of this cardiac channel. Identification of the transcription complexes, particularly those with cardiac specificity, that interact with these regions remains a major challenge, as does identification of more remote regions within the gene that also modulate expression. A polymorphism in a highly conserved region of the core promoter increased expression ~50% in neonatal myocytes. Given the evidence that loss of sodium channel function through any one of a number of mechanisms is arrhythmogenic, this polymorphism may represent the first example of an allele that could protect against serious arrhythmias, although extensive further studies in vitro, in vivo, and in clinical study populations will be required before this enters clinical practice. Cloning and initial characterization of this region will now allow us and others to proceed to further screening the region for DNA variants across populations and in particular in patients with serious arrhythmias.


   Acknowledgements
 
Supported in part by grants from the United States Public Health Service (HL46681, HL65962). Dr. Roden is the holder of the William Stokes Chair in Experimental Therapeutics, a gift from the Dai-ichi Corporation.


   Notes
 
Time for primary review 24 days

* The nucleotide sequence for the human hSCN5A promoter has been deposited in the GenBank database under GenBank accession number AY313163. The nucleotide sequence for the mouse mscn5a promoter has been deposited in the GenBank database under GenBank accession number AY313164. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Balser J.R. Inherited sodium channelopathies: models for acquired arrhythmias? Am. J. Physiol. Heart Circ. Physiol. (2002) 282:H1175–H1180.[Free Full Text]
  2. Roden D.M. The problem, challenge and opportunity of genetic heterogeneity in monogenic diseases predisposing to sudden death. J. Am. Coll. Cardiol. (2002) 40:357–359.[Free Full Text]
  3. Anderson M.E., Al Khatib S.M., Roden D.M., et al. Cardiac repolarization: current knowledge, critical gaps, and new approaches to drug development and patient management. Am. Heart J. (2002) 144:769–781.[CrossRef][Web of Science][Medline]
  4. Ruggieri V.L., Arberas C.L. Neuromuscular hereditary channelopathies: non-dystrophic myotonias, paramyotonias and periodic paralysis. Rev. Neurol. (2002) 34:150–156.[CrossRef][Web of Science][Medline]
  5. Bond E.F. Channelopathies: potassium-related periodic paralyses and similar disorders. AACN Clin. Issues (2000) 11:261–270.[CrossRef][Medline]
  6. Barchi R.L. Ion channel mutations affecting muscle and brain. Curr. Opin. Neurol. (1998) 11:461–468.[CrossRef][Web of Science][Medline]
  7. Ptacek L.J., Fu Y.H. Channelopathies: episodic disorders of the nervous system. Epilepsia (2002) 42(Suppl. 5):35–43.
  8. Huopio H., Shyng S.L., Otonkoski T., Nichols C.G. K(ATP) channels and insulin secretion disorders. Am. J. Physiol. Endocrinol. Metab. (2002) 283:E207–E216.[Abstract/Free Full Text]
  9. Miki T., Tashiro F., Iwanaga T., et al. Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc. Natl. Acad. Sci. U. S. A. (1997) 94:11969–11973.[Abstract/Free Full Text]
  10. Walder R.Y., Landau D., Meyer P., et al. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat. Genet. (2002) 31:171–174.[CrossRef][Web of Science][Medline]
  11. Snyder P.M. Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface. J. Clin. Invest. (2000) 105:45–53.[Web of Science][Medline]
  12. Ambrosius W.T., Bloem L.J., Zhou L., et al. Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension. Hypertension (1999) 34:631–637.[Abstract/Free Full Text]
  13. Goldin A.L., Barchi R.L., Caldwell J.H., et al. Nomenclature of voltage-gated sodium channels. Neuron (2000) 28:365–368.[CrossRef][Web of Science][Medline]
  14. Keating M.T., Sanguinetti M.C. Molecular and cellular mechanisms of cardiac arrhythmias. Cell (2001) 104:569–580.[CrossRef][Web of Science][Medline]
  15. The Cardiac Arrhythmia Suppression Trial II Investigators, Effect of the antiarrhythmic agent moricizine on survival after myocardial infarction. The cardiac arrhythmia suppression trial II investigators. N. Engl. J. Med. (1992) 327:227–233.[Abstract]
  16. Bigger J.T. Jr. Implications of the cardiac arrhythmia suppression trial for antiarrhythmic drug treatment. Am. J. Cardiol. (1990) 65:3D–10D.[Medline]
  17. Chen Q., Kirsch G.E., Zhang D., et al. Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature (1998) 392:293–296.[CrossRef][Medline]
  18. Zhang H., Maldonado M.N., Barchi R.L., Kallen R.G. Dual tandem promoter elements containing CCAC-like motifs from the tetrodotoxin-resistant voltage-sensitive Na+ channel (rSkM2) gene can independently drive muscle-specific transcription in L6 cells. Gene Expr. (1999) 8:85–103.[Web of Science][Medline]
  19. Sheng Z.H., Zhang H., Barchi R.L., Kallen R.G. Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter. DNA Cell Biol. (1994) 13:9–23.[Web of Science][Medline]
  20. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. Basic local alignment search tool. J. Mol. Biol. (1990) 215:403–410.[CrossRef][Web of Science][Medline]
  21. Wang Q., Li Z., Shen J., Keating M.T. Genomic organization of the human SCN5A gene encoding the cardiac sodium channel. Genomics (1996) 34:9–16.[CrossRef][Web of Science][Medline]
  22. Yang T., Kupershmidt S., Roden D.M. Anti-minK antisense decreases the amplitude of the rapidly activating cardiac delayed rectifier K+ current. Circ. Res. (1995) 77:1246–1253.[Abstract/Free Full Text]
  23. Kupershmidt S., Yang T., Chanthaphaychith S., Wang Z., Towbin J.A., Roden D.M. Defective human ether-a-go-go-related gene trafficking linked to an endoplasmic reticulum retention signal in the C terminus. J. Biol. Chem. (2002) 277:27442–27448.[Abstract/Free Full Text]
  24. Li H., Capetanaki Y. An E box in the desmin promoter cooperates with the E box and MEF-2 sites of a distal enhancer to direct muscle-specific transcription. EMBO J. (1994) 13:3580–3589.[Web of Science][Medline]
  25. Calvo S., Vullhorst D., Venepally P., Cheng J., Karavanova I., Buonanno A. Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription. Mol. Cell. Biol. (2001) 21:8490–8503.[Abstract/Free Full Text]
  26. Mitchell-Felton H., Hunter R.B., Stevenson E.J., Kandarian S.C. Identification of weight-bearing-responsive elements in the skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene. J. Biol. Chem. (2000) 275:23005–23011.[Abstract/Free Full Text]
  27. Bhavsar P.K., Dellow K.A., Yacoub M.H., Brand N.J., Barton P.J. Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. J. Mol. Cell. Cardiol. (2000) 32:95–108.[CrossRef][Web of Science][Medline]
  28. Esser K., Nelson T., Lupa-Kimball V., Blough E. The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transcription in skeletal muscle. J. Biol. Chem. (1999) 274:12095–12102.[Abstract/Free Full Text]
  29. Dellow K.A., Bhavsar P.K., Brand N.J., Barton P.J. Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter. Cardiovasc. Res. (2001) 50:24–33.[Abstract/Free Full Text]

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