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BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. We established the expression of seven main cardiac ion channel a-subunits and four b-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins

Background— Brugada syndrome is a heterogeneous heart rhythm disorder characterized by an atypical right bundle block pattern with ST-segment elevation and T-wave inversion in the right precordial leads. Loss-of-function mutations in SCN5A encoding the cardiac sodium channel Na V 1.5 are associated with Brugada syndrome. We found novel mutations in SCN5A in 2 different families diagnosed with Brugada syndrome and investigated how those affected Na V 1.5 channel function. Methods and Results— We performed genetic testing of the probands’ genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded for Na V 1.5 wild type and mutants heterologously expressed in Chinese hamster ovary-K1 cells. Proband 1 had two novel Na V 1.5 mutations: Na V 1.5-R811H and Na V 1.5-R620H. The Na V 1.5-R811H mutation showed a significant loss of function in peak Na + current density and alteration of biophysical kinetic parameters (inactivation and recovery from inactivation), whereas Na V 1.5-R620H had no significant effect on the current. Proband 2 had a novel Na V 1.5-S1218I mutation. Na V 1.5-S1218I had complete loss of function, and 1:1 expression of Na V 1.5-wild type and Na V 1.5-S1218I mimicking the heterozygous state revealed a 50% reduction in current compared with wild type, suggesting a functional haploinsufficiency in the patient. Conclusions— Na V 1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome.

Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-Src binds to the Cx43 scaffolding protein zonula occludens-1 (ZO-1) with a higher affinity than does Cx43. This suggests c-Src outcompetes Cx43 for binding to ZO-1, thus acting as a chaperone for ZO-1 and causing unhooking from Cx43. To determine whether c-Src/ZO-1 interactions affect Cx43 lateralization within the epicardial border zone, we performed Western blot, immunoprecipitation, and immunolocalization for active c-Src (p-cSrc) post-MI using a canine model of coronary occlusion. We found that post-MI p-cSrc interacts with ZO-1 as Cx43 begins to decrease its interaction with ZO-1 and undergo initial loss of intercalated disk localization. This indicates that the molecular mechanisms by which Cx43 is lost from the intercalated disk following MI includes an interaction of p-cSrc with ZO-1 and subsequent loss of scaffolding of Cx43 leaving Cx43 free to diffuse in myocyte membranes from areas of high Cx43, as at the intercalated disk, to regions of lower Cx43 content, the lateral myocyte membrane. Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart.