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Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for Ϸ75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding ␣1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTSsusceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane CaATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused Snitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.cardiac arrhythmia ͉ ion channel ͉ nitrosylation ͉ plasma membrane Ca-ATPase ͉ sodium current

Background-Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming ␣-subunit associated with 1 or more auxiliary ␤-subunits. Four different ␤-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome. Methods and Results-We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na v␤ -subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel ␣-subunit (hNa V 1.5). Compared with the wild-type, L179F-␤4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A ϩ WT-␤4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations. Conclusions-We provide the seminal report of SCN4B-encoded Na v␤ 4 as a novel LQT3-susceptibility gene. (Circulation.

Abstract-Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in Ϸ30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. SCN5A encodes the voltage-dependent sodium channel ␣-subunit protein SCN5A, also called hNa v 1.5, 1 found predominantly in human heart muscle. This channel is responsible for large peak inward sodium current (I Na ) that underlies excitability and conduction in working myocardium (atrial and ventricular cells) and special conduction tissue (Purkinje cells and others), and also for late I Na that influences repolarization and refractoriness. Three complete cDNA clones for this channel hH1, 2 hH1a, 3 and hH1b 4 differ in amino acid sequence in 5 of the 2016 positions (Table 1). In addition, these three clones differ from the deduced amino acid sequence for SCN5A obtained from the two human genome databases: Celera and the International Human Genome Sequencing Collaboration (IHGSC). Before the present study, it was not clear whether or not these differences are present in human population as common variants. From previous studies, we know that dramatic differences in current expression can be found when arrhythmia mutations are expressed in different background clones. 4 This study was designed to answer the questions: What is the common background sequence for SCN5A? Do common variations affect channel function? Does it matter which Na ϩ channel clone (ie, background sequence) is used for functional studies of wild-type and mutated channels? Materials and MethodsProtocols used in this investigation are more fully described in the expanded Materials and Methods section in the online data supplement available...

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Background and purpose: Fluoxetine (Prozac s ) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro. Experimental approach: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K þ and SCN5A Na þ channels heterologously expressed in HEK293 cells. Key results: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (I hERG ). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of I hERG but did not alter fluoxetine's effect on hERG channel protein trafficking. Conclusions and implications: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce I hERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Ye, Bin, Carmen R. Valdivia, Michael J. Ackerman, and Jonathan C. Makielski. A common human SCN5A polymorphism modifies expression of an arrhythmia causing mutation. Physiol Genomics 12: 187-193, 2003. First published November 26, 2002 10.1152 10. /physiolgenomics.00117. 2002 encodes the ␣-subunit of the ion channel that carries Na current in human heart. From a human cardiac cDNA library we recloned SCN5A. The new clone hH1b differed from existing clones hH1 in four and from hH1a in three positions. The common polymorphism H558R was uniquely present in hH1b. Voltage clamp study showed minor but potentially important kinetic differences between hH1b and the other clones. More dramatically, when the LQT3 mutation M1766L was introduced into the different clones, Na current was markedly reduced in the hH1 and hH1a backgrounds, whereas in hH1b the Na current was not reduced. Immunocytochemistry experiments showed a trafficking defect for M1766L Na channels in hH1 and hH1a but not in hH1b. The double-mutation M1766L/H558R in the hH1a background restored normal trafficking and current including persistent late current, suggesting the disease phenotype was the result of a "double hit" that included the common polymorphism, H558R. These results show that the choice of background clone must be carefully considered in mutagenesis studies. This also represents an example of intragenic complementation, the first for such a large protein. ion channels; trafficking defect; polymorphisms; LQT3; intragenic complementation THE HUMAN cardiac Na channel gene SCN5A encodes the ion channel hNa v 1.5 (9), which carries inward Na current (I Na ) in heart. Mutations in SCN5A cause arrhythmia syndromes including congenital long QT syndrome LQT3 and Brugada syndrome (2, 12). Two distinct full-length hNa v 1.5 clones isolated from human cardiac cDNA libraries, hH1 (8) and hH1a (10), have been used previously to characterize human I Na for wild-type channels and for channels containing putative arrhythmia-causing mutations. The sequences for hH1 and hH1a reportedly differ in 9 of the 2,016 amino acids (8, 10) and have never been studied under identical conditions. We hypothesized that these differences might affect function, either of wild-type channels, or when arrhythmia mutations were inserted into the different backgrounds. For this study, RT-PCR was used to obtain a third complete clone (designated hH1b) for hNa v 1.5. hH1b contained a known common polymorphism H558R whereby histidine (H) at position 558 is replaced by an arginine (R) (11, 17). We also studied an LQT3 mutation M1766L that was previously reported to have both decreased current expression and at the same time enhanced late current (relative to peak current) when expressed in the hH1a background (14). In the present study, I Na expression for the M1766L mutation expressed in the three background clones depended upon the polymorphism at position 558. Thus SCN5A serves as its own modifier gene. These results represent a novel form of "intragenic complementation" where a m...

The Na1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.