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      N-glycosylation–dependent regulation of hK 2P17.1 currents

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          Abstract

          Two pore-domain potassium (K 2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK 2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK 2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK 2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK 2P17.1. Here, we characterized glycosylation of hK 2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK 2P17.1 channels and channels lacking specific glycosylation sites were expressed in Xenopus laevis oocytes, HEK-293T cells, and HeLa cells. N-glycosylation was disrupted using N-glycosidase F and tunicamycin. hK 2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK 2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK 2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK 2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK 2P17.1 channel subunits and its pivotal role in cell-surface targeting. Our findings underline the functional relevance of N-glycosylation in biogenesis and membrane trafficking of ion channels.

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          Most cited references39

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            The SWISS-MODEL Repository and associated resources

            SWISS-MODEL Repository (http://swissmodel.expasy.org/repository/) is a database of 3D protein structure models generated by the SWISS-MODEL homology-modelling pipeline. The aim of the SWISS-MODEL Repository is to provide access to an up-to-date collection of annotated 3D protein models generated by automated homology modelling for all sequences in Swiss-Prot and for relevant models organisms. Regular updates ensure that target coverage is complete, that models are built using the most recent sequence and template structure databases, and that improvements in the underlying modelling pipeline are fully utilised. As of September 2008, the database contains 3.4 million entries for 2.7 million different protein sequences from the UniProt database. SWISS-MODEL Repository allows the users to assess the quality of the models in the database, search for alternative template structures, and to build models interactively via SWISS-MODEL Workspace (http://swissmodel.expasy.org/workspace/). Annotation of models with functional information and cross-linking with other databases such as the Protein Model Portal (http://www.proteinmodelportal.org) of the PSI Structural Genomics Knowledge Base facilitates the navigation between protein sequence and structure resources.
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              Molecular background of leak K+ currents: two-pore domain potassium channels.

              Two-pore domain K(+) (K(2P)) channels give rise to leak (also called background) K(+) currents. The well-known role of background K(+) currents is to stabilize the negative resting membrane potential and counterbalance depolarization. However, it has become apparent in the past decade (during the detailed examination of the cloned and corresponding native K(2P) channel types) that this primary hyperpolarizing action is not performed passively. The K(2P) channels are regulated by a wide variety of voltage-independent factors. Basic physicochemical parameters (e.g., pH, temperature, membrane stretch) and also several intracellular signaling pathways substantially and specifically modulate the different members of the six K(2P) channel subfamilies (TWIK, TREK, TASK, TALK, THIK, and TRESK). The deep implication in diverse physiological processes, the circumscribed expression pattern of the different channels, and the interesting pharmacological profile brought the K(2P) channel family into the spotlight. In this review, we focus on the physiological roles of K(2P) channels in the most extensively investigated cell types, with special emphasis on the molecular mechanisms of channel regulation.
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                Author and article information

                Contributors
                Role: Monitoring Editor
                Journal
                Mol Biol Cell
                Mol. Biol. Cell
                molbiolcell
                mbc
                mboc
                Molecular Biology of the Cell
                The American Society for Cell Biology
                1059-1524
                1939-4586
                01 June 2019
                : 30
                : 12
                : 1425-1436
                Affiliations
                [a ]Department of Cardiology, University of Heidelberg, 69120 Heidelberg, Germany
                [b ]DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University of Heidelberg, 69120 Heidelberg, Germany
                [c ]HCR, Heidelberg Center for Heart Rhythm Disorders, University of Heidelberg, 69120 Heidelberg, Germany
                [d ]Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Georg-August University of Göttingen, 37073 Göttingen, Germany
                [e ]DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, University of Göttingen, 37073 Göttingen, Germany
                University of Geneva
                Author notes
                *Address correspondence to: Constanze Schmidt ( Constanze.Schmidt@ 123456med.uni-heidelberg.de ).
                Article
                E18-10-0687
                10.1091/mbc.E18-10-0687
                6724686
                30969900
                2c6d371d-76f5-4629-bcdc-369c4f517571
                © 2019 Wiedmann et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

                This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.

                History
                : 31 October 2018
                : 01 March 2019
                : 03 April 2019
                Categories
                Articles
                Cell Physiology

                Molecular biology
                Molecular biology

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