RESUMO
Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.
Assuntos
Arritmias Cardíacas , Canal de Potássio KCNQ1 , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio ERG1/genética , Hibridização in Situ Fluorescente , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismoRESUMO
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.
Assuntos
Regulação para Baixo/fisiologia , Guanilato Quinases/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica/fisiologia , RatosRESUMO
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.
Assuntos
Átrios do Coração/citologia , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Análise de Variância , Animais , Fenômenos Biomecânicos , Western Blotting , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Etilmaleimida/farmacologia , Imunofluorescência , Integrina beta1/metabolismo , Masculino , Microscopia de Fluorescência , Modelos Biológicos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Proteínas SNARE/antagonistas & inibidores , Sarcolema/metabolismo , Resistência ao CisalhamentoRESUMO
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.
Assuntos
Arritmias Cardíacas , Canais de Potássio Éter-A-Go-Go , Canal de Potássio ERG1/genética , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Miócitos CardíacosRESUMO
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.
Assuntos
Cálcio , Calmodulina , Cálcio/metabolismo , Calmodulina/metabolismo , Adesão Celular , Distrofina/metabolismo , Adesões Focais/metabolismo , Glicoproteínas/metabolismo , Guanilato Quinases/química , Guanilato Quinases/metabolismo , Proteínas Quinases/metabolismo , Serina , Canais de Sódio/metabolismoRESUMO
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.