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N-terminal mutations in the KCNQ1 channel are frequently linked to fatal arrhythmias in newborn children and adolescents but the cellular mechanisms involved in this dramatic issue remain, however, to be discovered. Here, we analyzed the trafficking of a series of N-terminal truncation mutants and identified a critical trafficking motif of KCNQ1. This determinant is located in the juxtamembranous region preceding the first transmembrane domain of the protein. Three mutations (Y111C, L114P and P117L) implicated in inherited Romano-Ward LQT1 syndrome, are embedded within this domain. Reexpression studies in both COS-7 cells and cardiomyocytes showed that the mutant proteins fail to exit the endoplasmic reticulum. KCNQ1 subunits harboring Y111C or L114P exert a dominant negative effect on the wild-type KCNQ1 subunit by preventing plasma membrane trafficking of heteromultimeric channels. The P117L mutation had a less pronounced effect on the trafficking of heteromultimeric channels but altered the kinetics of the current. Furthermore, we showed that the trafficking determinant in KCNQ1 is structurally and functionally conserved in other KCNQ channels and constitutes a critical trafficking determinant of the KCNQ channel family. Computed structural predictions correlated the potential structural changes introduced by the mutations with impaired protein trafficking. In conclusion, our studies unveiled a new role of the N-terminus of KCNQ channels in their trafficking and its implication in severe forms of LQT1 syndrome.

The voltage-sensitive Na + channel Na v 1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3η, interacting with the Na v 1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Na v 1.5 both in COS-7 cells expressing recombinant Na v 1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Na v 1.5 colocalized at the intercalated discs. We tested the functional link between Na v 1.5 and 14-3-3η using the whole-cell patch-clamp configuration. Coexpressing Na v 1.5, the β1 subunit and 14-3-3η induced a negative shift in the inactivation curve of the Na + current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Na v 1.5 cytoplasmic I interdomain interacting with 14-3-3η. Reversal was also obtained with the dominant negative R56,60A 14-3-3η mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na + channel acting as a cofactor for the regulation of the cardiac Na + current.

Nearly a hundred different KCNQ1 mutations have been reported as leading to the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) regulates the KCNQ1–KCNE1 complex. In the present study, we show that PIP 2 affinity is reduced in three KCNQ1 mutant channels (R243H, R539W, and R555C) associated with the long QT syndrome. In giant excised patches, direct application of PIP 2 on the cytoplasmic face of the three mutant channels counterbalances the loss of function. Reintroduction of a positive charge by application of methanethiosulfonate ethylammonium on the cytoplasmic face of R555C mutant channels also restores channel activity. The channel affinity for a soluble analog of PIP 2 is decreased in the three mutant channels. By using a model that describes the KCNQ1–KCNE1 channel behavior and by fitting the relationship between the kinetics of deactivation and the current amplitude obtained in whole-cell experiments, we estimated the PIP 2 binding and dissociation rates on wild-type and mutant channels. The dissociation rate of the three mutants was higher than for the wild-type channel, suggesting a decreased affinity for PIP 2 . PIP 2 binding was magnesium-dependent, and the PIP 2 -dependent equilibrium constant in the absence of magnesium was higher with the wild-type than with the mutant channels. Altogether, our data suggest that a reduced PIP 2 affinity of KCNQ1 mutants can lead to the long QT syndrome.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits (“IonChips”) were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I to,f and I K,slow densities, and increased I Ks density in hypothyroid myocytes. In addition to effects on K + channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the α1C Ca 2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na + , Cl − , and inwardly rectifying K + channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel α- and β-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.