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International audience; Background: Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel.Objective: The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes.Methods: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions.Results: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex.Conclusion: This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Heterologously expressed hERG channels represent a mainstay of in vitro drug safety screens intended to mitigate risk of cardiac IKr block and sudden cardiac death. This is true even as more channel types are adopted as part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) intended to elevate specificity and thus enhance throughput of promising lead drugs. Until now, hERG1a homomeric channels have been used as a proxy for IKr despite a wealth of evidence showing that hERG1a/1b heteromers better represent native channels in terms of protein abundance and channel biophysical and pharmacological properties. Past efforts to create a stable hERG1a/1b cell line were met with unpredictable silencing of hERG1b expression despite stable integration of the gene into the HEK293 cell genome. Here we report a new cell line stably expressing hERG1a, with hERG1b reliably controlled by an inducible promoter sensitive to doxycycline. Co-immunoprecipitation, Western blot analysis and patch-clamp electrophysiology confirm the heteromeric composition of the expressed channels. Association with hERG1b was found to promote hERG1a protein levels and enhance membrane current levels. Optimal conditions for drug screening and experimental investigation were achieved at 24 h exposure to 100 ng/ml doxycycline. Differences in pharmacological sensitivity between homomeric and heteromeric channels were observed for dofetilide and ebastine, but not fluoxetine, as evaluated by their IC50 values. Using these values in the O'Hara-Rudy-CiPA in silico model revealed discrepancies in pro-arrhythmia risk, implying the hERG1a homomeric platform overestimates risk for these two drugs. Dofetilide block was use-dependent and faster for hERG1a/1b than hERG1a channels, whereas ebastine showed considerable block at rest and had a slower progression for hERG1a/1b channels. The hERG1a/1b cell line thus represents an advanced model for contemporary drug safety screening assays such as CiPA that employ IC50 values to estimate risk of proarrhythmia in computational models of ventricular cardiomyocytes. This novel technology fulfills an unmet need to enhance specificity and foster a safe yet expanded drug development pipeline.

Membrane-associated guanylate kinase (MAGUK) proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein CASK (Calcium/CAlmodulin-dependent Serine protein Kinase) negatively regulates the main cardiac sodium channel, NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex, and like syntrophin, binds to the C-terminal domain of the channel. Here we show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Thus, the multi-modular structure of CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocyte.SUMMARYSequential functional domain deletion approach identifies three critical domains of CASK in cardiomyocytes. CASK binds the cell adhesion dystrophin-glycoprotein complex through HOOK domain and inhibits NaV1.5 channel membrane expression by impeding trafficking through L27B and GUK domains.

Catastrophic arrhythmias and sudden cardiac death can occur with even a small imbalance between inward sodium currents and outward potassium currents, but mechanisms establishing this critical balance are not understood. Here, we show that mRNA transcripts encoding INa and IKr channels (SCN5A and hERG, respectively) are associated in defined complexes during protein translation. Using biochemical, electrophysiological and single-molecule fluorescence localization approaches, we find that roughly half the hERG translational complexes contain SCN5A transcripts. Moreover, the transcripts are regulated in a way that alters functional expression of both channels at the membrane. Association and coordinate regulation of transcripts in discrete ‘microtranslatomes’ represents a new paradigm controlling electrical activity in heart and other excitable tissues.

Activation of the electrical signal and its transmission as a depolarizing wave in the whole heart requires highly organized myocyte architecture and cell-cell contacts. In addition, complex trafficking and anchoring intracellular machineries regulate the proper surface expression of channels and their targeting to distinct membrane domains. An increasing list of proteins, lipids, and second messengers can contribute to the normal targeting of ion channels in cardiac myocytes. However, their precise roles in the electrophysiology of the heart are far from been extensively understood. Nowadays, much effort in the field focuses on understanding the mechanisms that regulate ion channel targeting to sarcolemma microdomains and their organization into macromolecular complexes. The purpose of the present section is to provide an overview of the characterized partners of the main cardiac sodium channel, NaV1.5, involved in regulating the functional expression of this channel both in terms of trafficking and targeting into microdomains.

Rationale: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, Na V 1.5, carries the sodium current ( I Na ) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, Na V 1.5 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 Na V 1.5 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 I Na in vitro. In vivo CASK knockdown similarly increased I Na recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of Na V 1.5. CASK silencing reduces syntrophin expression without affecting Na V 1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of Na V 1.5 without changing mRNA levels. Quantification of Na V 1.5 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 I Na 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 Na V 1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

Introduction: Proteins of the MAGUK family have emerged as key components in ion channels organization and regulation within specialized submembrane domains of cardiomyocytes. In this context, we investigated for the first time the expression, localization and function in the heart of the MAGUK protein CASK. Methods and Results: Surprisingly, no signal was detectable for CASK at intercalated discs, making CASK the first MAGUK to be excluded from these structures. On the contrary, CASK was located at the lateral membrane where it belonged specifically to the costameric dystrophin/glycoproteins complex (DGC). Since a lateral sub-population of Nav1.5 channels have been reported to interact with syntrophin, a member of DGC, we hypothesized that CASK could modulate Nav1.5 channels. Using high resolution 3-D deconvolution microscopy, we observed a co-localization of CASK and Nav1.5 in cardiomyocytes and co-IP experiments confirmed that the two proteins are in the same complex. Whole-cell patch-clamp recordings revealed a negative regulation by CASK of the sodium current INa carried by Nav1.5 channels in cardiomyocytes. Using super resolution scanning coupled to conventional cell-attached patch-clamp, we investigated the involvement of CASK on Nav1.5 function and localization in highly confined microdomains of the lateral membrane. INa was recorded in crests and T-tubules on freshly isolated cardiomyocytes obtained from adult rats injected with Adeno Associated Virus (eGFP-AAV-ShCASK or eGFP-AAV-ShScr). CASK silencing caused a drastic reduction of INa at the crest whereas it increased the current at the T-tubules suggesting that CASK retains Nav1.5 channels at the crest and prevents their clustering in T-tubules. In vivo CASK silencing in rat using an eGFP-AAV-ShCASK-injected was associated with a conduction slowing as indicated by the prolongation of the QRS duration, cardiac dilation and left ventricle systolic dysfunction. Conclusion: Taken together these results indicate that CASK is a major determinant of the organization of a subpopulation of Nav1.5 at the costamere of cardiomyocytes and that CASK-silencing induces electrical and morphological remodeling.

Trafficking of ion channels in cardiomyocytes is a highly dynamic process which is determinant for shaping action potential (AP). In this study, we focused on the dynamic of Kv1.5 channel which carry the atria-specific potassium outward current involved in the repolarisation of the AP. We first investigated the endocytosis pathway for Kv1.5 channels in the atria. High resolution 3-D microscopy revealed that Kv1.5 channels are associated with clathrin vesicles in atrial myocytes but not with caveolin. Electron microscopy showed that vesicles are found both at the lateral sarcolemma and at the intercalated disc. Blockade of the clathrin pathway using sucrose or SiRNA induced an increase in IKur recorded by whole-cell patch-clamp and an accumulation of Kv1.5 channels at the sarcolemma as shown by biotinylation assay. Clathrin blockade also increased fluorescence recovery after photo bleaching of Kv1.5 channels. Altogether, these data show that Kv1.5 channels are internalized through the clathrin pathway. TIRF microscopy approach was used to follow eGFPKv1.5-Kv1.5 channels in living cells and to establish their dynamic in control and clathrin-blocked myocytes. Blockade of the clathrin pathway leads to a global increase of Kv1.5 channels at the membrane. Particle analysis revealed an accumulation of the channels into clusters and their stabilisation at the membrane. Our objective now is to investigate the involvement of the cytoskeleton in Kv1.5 channels endocytosis by using dyes staining tubulin or actin in live cells; and the fate of internalized channels at different time points by co-immunostainings with antibodies directed against the different endosomes. In conclusion, we have identified the clathrin pathway as the internalization route for Kv1.5 channel, in atrial myocytes. The blockade of this pathway modifies Kv1.5 channels dynamic at the membrane. Future work will be conducted to further investigate dynamic and fate of this atria-specific channel. This study should help understanding the constitution of the sub-membrane reservoir of repolarization that we previously described (Balse E., et al. 2009, PNAS).

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.

Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.

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.