Expression and intracellular localization of an SCN5A double mutant R1232W/T1620M implicated in Brugada syndrome.

Brugada syndrome is an inherited cardiac disorder caused by mutations in the cardiac sodium channel gene, SCN5A, that leads to ventricular fibrillation and sudden death. This study reports the changes in functional expression and cellular localization of an SCN5A double mutant (R1232W/T1620M) recently discovered in patients with Brugada syndrome. Mutant and wild-type (WT) human heart sodium channels (hNa(v)1.5) were expressed in tsA201 cells in the presence of the beta(1)-auxiliary subunit. Patch-clamp experiments in whole-cell configuration were conducted to assess functional expression. Immunohistochemistry and confocal microscopy were used to determine the spatial distribution of either WT or mutant cardiac sodium channels. The results show an abolition of functional sodium channel expression of the hNa(v)1.5/R1232W/T1620M mutant in the tsA201 cells. A conservative positively charged mutant, hNa(v)1.5/R1232K/T1620M, produced functional channels. Immunofluorescent staining showed that the FLAG-tagged hNa(v)1.5/WT transfected into tsA201 cells was localized on the cell surface, whereas the FLAG-tagged hNa(v)1.5/R1232W/T1620M mutant was colocalized with calnexin within the endoplasmic reticulum (ER). These results indicate that a positively charged arginine or lysine residue at position 1232 in the double mutant is required for the proper transport and functional expression of the hNa(v)1.5 protein. These results support the concept that loss of function of the cardiac Na(+) channel is responsible for the Brugada syndrome. The full text of this article is available at http://www.circresaha.org.

A novel SCN5A mutation manifests as a malignant form of long QT syndrome with perinatal onset of tachycardia/bradycardia.

OBJECTIVE: Congenital long QT syndrome (LQTS) with in utero onset of the rhythm disturbances is associated with a poor prognosis. In this study we investigated a newborn patient with fetal bradycardia, 2:1 atrioventricular block and ventricular tachycardia soon after birth. METHODS: Mutational analysis and DNA sequencing were conducted in a newborn. The 2:1 atrioventricular block improved to 1:1 conduction only after intravenous lidocaine infusion or a high dose of mexiletine, which also controlled the ventricular tachycardia. RESULTS: A novel, spontaneous LQTS-3 mutation was identified in the transmembrane segment 6 of domain IV of the Na(v)1.5 cardiac sodium channel, with a G-->A substitution at codon 1763, which changed a valine (GTG) to a methionine (ATG). The proband was heterozygous but the mutation was absent in the parents and the sister. Expression of this mutant channel in tsA201 mammalian cells by site-directed mutagenesis revealed a persistent tetrodotoxin-sensitive but lidocaine-resistant current that was associated with a positive shift of the steady-state inactivation curve, steeper activation curve and faster recovery from inactivation. We also found a similar electrophysiological profile for the neighboring V1764M mutant. But, the other neighboring I1762A mutant had no persistent current and was still associated with a positive shift of inactivation. CONCLUSIONS: These findings suggest that the Na(v)1.5/V1763M channel dysfunction and possible neighboring mutants contribute to a persistent inward current due to altered inactivation kinetics and clinically congenital LQTS with perinatal onset of arrhythmias that responded to lidocaine and mexiletine.

The C-terminal region as a modulator of rNa(v)1.7 and rNa(v)1.8 expression levels.

Mammalian cells poorly express rNa(v)1.8 channels. In contrast, rNa(v)1.7 dorsal root ganglion channels have 90-fold higher peak Na(+) current densities. We investigated the role of rNa(v)1.7 and rNa(v)1.8 carboxy-termini in modulating the expression of rNa(v)1.7 and rNa(v)1.8 channels in tsA201 cells. Mutations in the ubiquitination site of the C-terminus did not improve rNa(v)1.8 current levels. However, rNa(v)1.8 chimeras containing the entire or the proximal portion of the rNa(v)1.7 C-terminus expressed 3.2-fold and 4.8-fold higher peak current densities, respectively, than parent rNa(v)1.8 channels. We conclude that the two Na(+) channels may have different endoplasmic reticulum processing signals.

Biophysical characteristics of a new mutation on the KCNQ1 potassium channel (L251P) causing long QT syndrome.

The congenital long QT syndrome (LQTS) is a hereditary cardiac disease characterized by prolonged ventricular repolarization, syncope, and sudden death. Mutations causing LQTS have been identified in various genes that encode for ionic channels or their regulatory subunits. Several of these mutations have been reported on the KCNQ1 gene encoding for a potassium channel or its regulatory subunit (KCNE1). In this study, we report the biophysical characteristics of a new mutation (L251P) in the transmembrane segment 5 (S5) of the KCNQ1 potassium channel. Potassium currents were recorded from CHO cells transfected with either wild type or mutant KCNQ1 in the presence or in the absence of its regulatory subunit (KCNE1), using the whole-cell configuration of the patch clamp technique. Wild-type KCNQ1 current amplitudes are increased particularly by KCNE1 co-expression but no current is observed with the KCNQ1 (L251P) mutant either in the presence or in the absence of KCNE1. Coexpressing KCNE1 with equal amount of cDNAs encoding wild type and mutant KCNQ1 results in an 11-fold reduction in the amplitude of potassium currents. The kinetics of activation and inactivation and the activation curve are minimally affected by this mutation. Our results suggest that the dominant negative effect of the P251L mutation on KCNQ1 channel explains the prolonged repolarization in patients carrying this mutation.

A novel mutation in SCN5A, delQKP 1507-1509, causing long QT syndrome: role of Q1507 residue in sodium channel inactivation.

Inherited long QT syndrome (LQTS) is caused by mutations in six genes including SCN5A, encoding the alpha-subunit of the human cardiac voltage-dependent sodium channel hNa(v)1.5. In LQT3, various mutations in SCN5A were identified, which produce a gain of channel function. The aim of this study was to screen SCN5A for mutations in a family with the LQT3 phenotype and to analyze the consequences of the mutation on the channel function. By polymerase chain reaction-denaturating high performance liquid chromatography-sequencing, we identified a novel deletion in SCN5A, delQKP 1507-1509, in the DIII-DIV linker of the sodium channel. The hNa(v)1.5/delQKP1507-1509, hNa(v)1.5/delQ1507 and hNa(v)1.5/Q1507A mutants were constructed in vitro, mutant channels were expressed in the tsA201 human cell line and studied using the whole-cell configuration of the patch clamp technique. A persistent inward sodium current of 1-1.5% of maximum currents measured at -30 mV in all mutant sodium channels was recorded, which was nearly completely blocked by the sodium-channel blockers tetrodotoxin and lidocaine. The deletion mutants resulted in a significant shift of steady-state activation to more depolarized voltages. The delQ1507 showed a small shift of steady-state inactivation towards more negative potentials, whereas no significant shifts were observed in both steady-state activation and inactivation in Q1507A compared to the wild-type Na(v)1.5 sodium channels. The novel SCN5A mutation, delQKP, induces a residual current as previously shown for other SCN5A mutations causing LQTS. DelQKP shares the deletion of Q1507 with the formerly known delKPQ 1505-1507. Our data suggest that Q1507 plays an important role in fast sodium channel inactivation.