Lateral Membrane-Specific MAGUK CASK Down-Regulates NaV1.5 Channel in Cardiac Myocytes.

RATIONALE: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. OBJECTIVE: To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. METHODS AND RESULTS: Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. CONCLUSION: This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

A truncating SCN5A mutation combined with genetic variability causes sick sinus syndrome and early atrial fibrillation.

BACKGROUND: Mutations in the SCN5A gene, encoding the α subunit of the cardiac Na(+) channel, Nav1.5, can result in several life-threatening arrhythmias. OBJECTIVE: To characterize a distal truncating SCN5A mutation, R1860Gfs*12, identified in a family with different phenotypes including sick sinus syndrome, atrial fibrillation (AF), atrial flutter, and atrioventricular block. METHODS: Patch-clamp and biochemical analyses were performed in human embryonic kidney 293 cells transfected with wild-type (WT) and/or mutant channels. RESULTS: The mutant channel expressed alone caused a 70% reduction in inward sodium current (INa) density compared to WT currents, which was consistent with its partial proteasomal degradation. It also led to a negative shift of steady-state inactivation and to a persistent current. When mimicking the heterozygous state of the patients by coexpressing WT and R1860Gfs*12 channels, the biophysical properties of INa were still altered and the mutant channel α subunits still interacted with the WT channels. Since the proband developed paroxysmal AF at a young age, we screened 17 polymorphisms associated with AF risk in this family and showed that the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF development. In addition, when mimicking the difference in resting membrane potentials between cardiac atria and ventricles in human embryonic kidney 293 cells or when using computer model simulations, R1860Gfs*12 induced a more drastic decrease in INa at the atrial potential. CONCLUSION: We have identified a distal truncated SCN5A mutant associated with gain- and loss-of-function effects, leading to sick sinus syndrome and atrial arrhythmias. A constitutively higher susceptibility to arrhythmias of atrial tissues and genetic variability could explain the complex phenotype observed in this family.