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The density of functional Kv1.5 channels underlying IKur, the main repolarizing current in human atria, is a result of the equilibrium between exocytosis and endocytosis. We have shown that in addition to constitutive exocytosis, Kv1.5 channels can also undergo regulated exocytosis, for instance following changes in the mechanical environment or changes in the cholesterol content of the sarcolemma. Although constitutive and triggered endocytosis have been investigated for Kv1.5, its internalization pathway has not been described. Using high-resolution 3-D deconvolution microscopy, we showed that Kv1.5 channels are associated with clathrin vesicles (CVs) in atrial myocytes. Electron microscopy (EM) showed that CVs are found both at the intercalated disc and at the lateral sarcolemma, aligned along z-bands. Blockade of the clathrin pathway using hypertonic media or SiRNA increased IKur densityin atrial myocytes and led to Kv1.5 channels accumulating at the sarcolemma, as shown by biotinylation assays and fluorescence recovery after photobleaching (FRAP) experiments. These data support the hypothesis that Kv1.5 channels are internalized via the clathrin pathway.Next, we investigated Kv1.5 channel internalization in a rat model of atrial hemodynamic overload. Despite reduced Kv1.5 protein expression in dilated atria, IKur density was unchanged, suggesting increased functional Kv1.5 channels at the sarcolemma. Clathrin expression was reduced in dilated atria, and a decreased colocalization between Kv1.5 channels and CVs was observed. However, EM showed no significant difference in internalization activity between sham and dilated atria. Therefore, the reduced clathrin protein synthesis observed in dilated atria is not likely to be responsible for the accumulation of Kv1.5 channels at the sarcolemma. Other mechanisms such as increased recycling and/or membrane stabilization must be investigated to understand how IKur is maintained in dilated atria.

The Shal-type Kv channels (Kv4.x) account for a large part of the outward potassium current, Ito, in heart. Membrane-associated guanylate kinase proteins are major determinants of the organization of several ion channels however, few are known on the interaction between Kv4.x channels and cardiac MAGUK, SAP97 in the heart. Here using pulldown assays we found a direct interaction via the VSAL amino acid motif between the Kv4.x C-terminus and the SAP97 in rat and human myocardia. In Kv4.3-KChIP stable CHO cell line and using the whole cell patch clamp technique, SAP97 increased the Kv4.3 encoded current by a factor 2 (145 ± 19pA/pF vs 300 ± 52 pA/pF; n=11; p

We have previously shown that in the rat the sarco/endoplasmic reticulum calcium ATPase, SERCA2a, controls vascular smooth muscle cell (VSMC) proliferation through modulation of the activity of the transcription factor NFAT. Here we tested the hypothesis that SERCA2a, controls human VSMC proliferation by inhibiting voltage-independent Ca2+ entry and the NFAT transcription pathway.Single-channel recording showed that proliferating VSMC possessed high spontaneous voltage-independent basal channel activity (nPo: 0.415 ± 0.050; n=31). SERCA2a gene transfer, using an adenoviral vector, to proliferating VSMCs abolished the activity of these channels (0.091 ± 0.096; n=26) compared to control cells infected with an adenovirus encoding GFP (0.315 ± 0.035; n=42). SERCA 2a gene transfer also down-regulated expression of transient receptor potential channels TRPC4, TRPC5 and stromal interacting molecule 1 (STIM1) suggesting their involvement in the channel activity.SERCA2a gene transfer also inhibited VSMC proliferation and migration as well as the NFAT activity. Furthermore, NFAT activity was inhibited by depolarization-induced Ca2+ influx and by several calcium channel inhibitors such as nifedipine, mibefradil, carboxyamidotriazole and 2-aminoethoxydiphenyl, suggesting involvement of different types of Ca2+ channels in the control of NFAT and proliferation. Our data suggest that different types of Ca2+ channels are involved in the control of NFAT transcriptional activity and proliferation. By controlling submembrane Ca2+ concentration, SERCA2a finely regulated basal Ca2+ current via regulation of translocation and/or expression of STIM1 and TRPCs.

During the cardiac cycle atrial myocytes are continuously exposed to shear stress, which occurs when laminar sheets of cells move relative to each other. However, in atrial myocytes the effects of shear stress on ion channel regulation are poorly known. Here, we report that shear stress activates a large outward current, and corresponding decreased action potential duration in atrial myocytes, primarily attributable to an increase in the membrane density of the potassium channel Kv1.5. Total outward currents were reversibly increased upon shear stress from 3.8 ± 0.5 pA/pF to 43.2 ± 8.9 pA/pF, and could be blocked using 1mM 4-AP. The increase in current was due to recruitment of channels from submembranous pools to the plasma membrane, as it could be prevented by the SNARE protein inhibitor NEM, as well as with BAPTA to buffer intracellular calcium. Recruitment of EGFP-Kv1.5 could be directly observed using total internal reflection fluorescence microscopy. The mechanosensor was found to be the integrin system signalling through focal adhesion kinase, and an intact microtubule system was required. In a rat model of heart failure (HF), the integrin system was upregulated and myocytes showed an increase in basal current. Furthermore, HF myocytes had reduced Kv1.5 expression as well as a reduced response to shear stress. These data suggest that under conditions in which the integrin system is altered, the signalling pathway resulting in recruitment of Kv1.5 is overactivated leading to an increase in basal current and a reduced capacity to respond to further stimulation. We propose that pools of Kv1.5 comprise an inducible reserve which can mediate the repolarization of the atrium following mechanical stress, and that this system is disrupted when the mechanosensor is altered.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers which regulate many biological processes. They can be eliminated by active efflux transporters, namely the multidrug resistance proteins MRP4 and MRP5. To delineate the role of MRP4/5, we studied arterial smooth muscle cells in which the role of cyclic nucleotide levels on proliferation has been well-established.Methods: Human SMCs were isolated from the coronary artery media from patients. Small interfering RNAs (siRNA) specific for MRP4 mRNA were designed and validated. Adenovirus encoding MRP4 short hairpin RNA (Ad-shMRP4) were used for in vivo studies.Results: MRP4 was over-expressed in serum-induced proliferating SMC as well as in atherosclerotic plaques in human coronary arteries and in neo-intima of injured rat carotid arteries. Inhibition of MRP4 by siRNA blocked VSMC proliferation in vitro. In balloon-injured rat carotid arteries, intima/media ratios were significantly lower in Ad-shMRP4-infected arteries than in Ad-shLuciferase-infected arteries (0.65 ± 0.1 vs 1.05 ± 0.2; p

Heterozygous loss-of-function mutations in the SCN5A gene encoding the cardiac Na+ channel Nav1.5 cause various hereditary arrhythmias including Brugada syndrome (BrS), a disease characterized by ST-segment elevation in the ECG right precordial leads, and an increased risk of sudden cardiac death. Here, we aimed to study the role of the Nav1.5 evolutionarily conserved N-terminus of which the function remains unknown. We characterized two mutations within the cytoplasmic N-terminus of Nav1.5, R104W and R121W identified in BrS patients and a construct where this region was deleted. In HEK cells, R104W, R121W and Nav1.5-ΔNter abolished INa. Immunostaining of Nav1.5 transfected in neonatal rat cardiomyocytes showed retention of R104W and R121W in cytoplasmic compartments, as opposed to the WT channels. Moreover, Western blot analysis revealed that mutants were mostly degraded and that the ubiquitin proteasome inhibitor, MG132, prevented this degradation. Coexpression of WT with either mutants, mimicking the heterozygous state of BrS patients, led to a marked INa density reduction (80%) and a 8 mV positive shift of the V0.5 activation, compared to WT alone, demonstrating a dominant-negative effect. Interestingly, when Nav1.5-ΔNter was coexpressed with WT, no dominant-negative effect was observed. We demonstrated that (i) the two N-terminal mutation abolish INa through retention of the mutant channels and their subsequent degradation by the proteasome, (ii) the two mutant channels exert a specific dominant-negative effect on WT channels by producing a default in WT channel trafficking to the membrane, (iii) these mutations have more drastic effects on INa than deletion of this region. Altogether, our results suggest that the N-terminal domain of Nav1.5 plays an important role in the traffic of Nav1.5 subunits to the plasma membrane, the regulation of their expression and their activation.