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Christensen AH, Pan G, Marvig RL, Rodriguez Gonzalez FG, Vissing CR, Silajdzija E, Frosted R, Girma EG, Gabrielaite M, Jensen HK, Rossing K, Henriksen FL, Sandgaard NCF, Ahlberg G, Ghouse J, Lundegaard PR, Weischenfeldt J, Wadelius C, Bundgaard H. Gain-of-function enhancer variant near KCNB1 causes familial ST-depression syndrome. Eur Heart J 2025:ehaf213. [PMID: 40208226 DOI: 10.1093/eurheartj/ehaf213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2024] [Revised: 01/06/2025] [Accepted: 03/17/2025] [Indexed: 04/11/2025] Open
Abstract
BACKGROUND AND AIMS Familial ST-depression syndrome (FSTD) is a recently identified inherited cardiac disease associated with arrhythmias and systolic dysfunction. The underlying genetic aetiology has remained elusive. This study aimed at finding the causative variant. METHODS A total of 67 FSTD patients (20 families) were studied. Linkage analysis and whole-genome sequencing (WGS) were initially performed. An identified non-coding variant was functionally characterized in AC16 human cardiomyocytes, muscle tissue, and human myocardium. In silico analyses, luciferase and dCas9-activator/repressor assays, protein-DNA experiments, chromosome conformation capture (4C), and RNA sequencing were also performed. RESULTS The electrocardiographic (ECG) phenotype was inherited in an autosomal dominant manner in all families. Linkage analysis revealed a single peak on chromosome 20, and WGS identified a single, rare, non-coding variant located 18 kb downstream of KCNB1 on chromosome 20 in all affected individuals. Perfect co-segregation with the ECG phenotype was observed together with full penetrance in all families. The variant creates a MEF2-binding site and presence of the variant allele or MEF2 co-expression enhanced transcriptional activity. dCas9-activator/repressor assays showed that KCNB1 was the only gene consistently regulated by the locus and 4C experiments in AC16 cells and human muscle tissue confirmed the locus-KCNB1 promoter interaction. Expression analysis in human endocardial tissue did not document any change in gene expression likely explained by expressional heterogeneity. CONCLUSIONS A gain-of-function enhancer variant creates a hyperactive regulatory locus that interacts with the KCNB1 promoter and causes FSTD. This is the first time that KCNB1 has been implicated in human cardiac electrophysiology and arrhythmogenesis.
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Affiliation(s)
- Alex Hørby Christensen
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
- Department of Cardiology, Copenhagen University Hospital-Herlev-Gentofte Hospital, Borgmester Ib Juuls Vej 1, DK-2730 Herlev, Denmark
| | - Gang Pan
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
| | - Rasmus L Marvig
- Department of Genomic Medicine, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark
| | - Francisco German Rodriguez Gonzalez
- Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
- Finsen Laboratory, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark
| | - Christoffer Rasmus Vissing
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
| | - Elvira Silajdzija
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
| | - Rasmus Frosted
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
- Department of Cardiology, Copenhagen University Hospital-Herlev-Gentofte Hospital, Borgmester Ib Juuls Vej 1, DK-2730 Herlev, Denmark
| | - Etsehiwot Girum Girma
- Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
- Finsen Laboratory, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark
| | - Migle Gabrielaite
- Department of Genomic Medicine, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark
| | - Henrik Kjærulf Jensen
- Department of Cardiology, Aarhus University Hospital, Aarhus, Denmark
- Department of Clinical Medicine, Health, Aarhus University, Aarhus, Denmark
- European Reference Network for Rare, Low Prevalence and Complex Diseases of the Heart: ERN GUARD-Heart
| | - Kasper Rossing
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
| | | | - Niels Christian Foldager Sandgaard
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
- Department of Cardiology, Odense University Hospital, Odense, Denmark
| | - Gustav Ahlberg
- Laboratory for Molecular Cardiology, The Heart Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - Jonas Ghouse
- Laboratory for Molecular Cardiology, The Heart Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
- Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark
| | | | - Joachim Weischenfeldt
- Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
- Finsen Laboratory, Copenhagen University Hospital-Rigshospitalet, Copenhagen, Denmark
| | - Claes Wadelius
- Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
| | - Henning Bundgaard
- The Unit for Inherited Cardiac Diseases, Department of Cardiology Section 2142, The Heart Centre, Copenhagen University Hospital-Rigshospitalet, Inge Lehmanns Vej 7, DK-2100 Copenhagen OE, Denmark
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Knaus LS, Basilico B, Malzl D, Gerykova Bujalkova M, Smogavec M, Schwarz LA, Gorkiewicz S, Amberg N, Pauler FM, Knittl-Frank C, Tassinari M, Maulide N, Rülicke T, Menche J, Hippenmeyer S, Novarino G. Large neutral amino acid levels tune perinatal neuronal excitability and survival. Cell 2023; 186:1950-1967.e25. [PMID: 36996814 DOI: 10.1016/j.cell.2023.02.037] [Citation(s) in RCA: 26] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 02/03/2023] [Accepted: 02/23/2023] [Indexed: 03/31/2023]
Abstract
Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.
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Affiliation(s)
- Lisa S Knaus
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Bernadette Basilico
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Daniel Malzl
- Max Perutz Labs, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
| | - Maria Gerykova Bujalkova
- Institute of Medical Genetics, Medical University of Vienna, Währinger Straße 10, 1090 Vienna, Austria
| | - Mateja Smogavec
- Institute of Medical Genetics, Medical University of Vienna, Währinger Straße 10, 1090 Vienna, Austria
| | - Lena A Schwarz
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Sarah Gorkiewicz
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Nicole Amberg
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Florian M Pauler
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Christian Knittl-Frank
- Institute of Organic Chemistry, University of Vienna, Währinger Strasse 38, 1090 Vienna, Austria
| | - Marianna Tassinari
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Nuno Maulide
- Institute of Organic Chemistry, University of Vienna, Währinger Strasse 38, 1090 Vienna, Austria; University of Vienna, Research Platform NeGeMac, Währinger Strasse 38, 1090 Vienna, Austria
| | - Thomas Rülicke
- University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
| | - Jörg Menche
- Max Perutz Labs, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
| | - Simon Hippenmeyer
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria
| | - Gaia Novarino
- Institute of Science and Technology Austria (ISTA), Am Campus 1, 3400 Klosterneuburg, Austria.
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Khvotchev M, Soloviev M. SNARE Modulators and SNARE Mimetic Peptides. Biomolecules 2022; 12:biom12121779. [PMID: 36551207 PMCID: PMC9776023 DOI: 10.3390/biom12121779] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Revised: 11/22/2022] [Accepted: 11/26/2022] [Indexed: 12/03/2022] Open
Abstract
The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.
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Affiliation(s)
- Mikhail Khvotchev
- Department of Biochemistry, Center for Neuroscience, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
- Correspondence: (M.K.); (M.S.)
| | - Mikhail Soloviev
- Department of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK
- Correspondence: (M.K.); (M.S.)
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Stožer A, Paradiž Leitgeb E, Pohorec V, Dolenšek J, Križančić Bombek L, Gosak M, Skelin Klemen M. The Role of cAMP in Beta Cell Stimulus-Secretion and Intercellular Coupling. Cells 2021; 10:1658. [PMID: 34359828 PMCID: PMC8304079 DOI: 10.3390/cells10071658] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 06/18/2021] [Accepted: 06/28/2021] [Indexed: 12/22/2022] Open
Abstract
Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus-secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca2+ signal and the most distal exocytosis. In addition to modifying intracellular coupling, the effect of cAMP on insulin secretion could also be at least partly explained by the impact on intercellular coupling. In this review, we systematically describe the possible roles of cAMP at these intra- and inter-cellular signaling nodes, keeping in mind the relevance for the whole organism and translation to humans.
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Affiliation(s)
- Andraž Stožer
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
| | - Eva Paradiž Leitgeb
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
| | - Viljem Pohorec
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
| | - Jurij Dolenšek
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
- Faculty of Natural Sciences and Mathematics, University of Maribor, SI-2000 Maribor, Slovenia
| | - Lidija Križančić Bombek
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
| | - Marko Gosak
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
- Faculty of Natural Sciences and Mathematics, University of Maribor, SI-2000 Maribor, Slovenia
| | - Maša Skelin Klemen
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000 Maribor, Slovenia; (A.S.); (E.P.L.); (V.P.); (J.D.); (L.K.B.); (M.G.)
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Clenbuterol-sensitive delayed outward potassium currents in a cell model of spinal and bulbar muscular atrophy. Pflugers Arch 2021; 473:1213-1227. [PMID: 34021780 DOI: 10.1007/s00424-021-02559-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2021] [Revised: 03/09/2021] [Accepted: 03/23/2021] [Indexed: 10/21/2022]
Abstract
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. SBMA is characterized by selective dysfunction and degeneration of motor neurons in the brainstem and spinal cord through still unclear mechanisms in which ion channel modulation might play a central role as for other neurodegenerative diseases. The beta2-adrenergic agonist clenbuterol was observed to ameliorate the SBMA phenotype in mice and patient-derived myotubes. However, the underlying molecular mechanism has yet to be clarified. Here, we unveil that ionic current alterations induced by the expression of polyQ-expanded AR in motor neuron-derived MN-1 cells are attenuated by the administration of clenbuterol. Our combined electrophysiological and pharmacological approach allowed us to reveal that clenbuterol modifies delayed outward potassium currents. Overall, we demonstrated that the protection provided by clenbuterol restores the normal function through the modulation of KV2-type outward potassium currents, possibly contributing to the protective effect on motor neuron toxicity in SBMA.
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6
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Hewlett B, Singh NP, Vannier C, Galli T. ER-PM Contact Sites - SNARING Actors in Emerging Functions. Front Cell Dev Biol 2021; 9:635518. [PMID: 33681218 PMCID: PMC7928305 DOI: 10.3389/fcell.2021.635518] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Accepted: 01/21/2021] [Indexed: 11/13/2022] Open
Abstract
The compartmentalisation achieved by confining cytoplasm into membrane-enclosed organelles in eukaryotic cells is essential for maintaining vital functions including ATP production, synthetic and degradative pathways. While intracellular organelles are highly specialised in these functions, the restricting membranes also impede exchange of molecules responsible for the synchronised and responsive cellular activities. The initial identification of contact sites between the ER and plasma membrane (PM) provided a potential candidate structure for communication between organelles without mixing by fusion. Over the past decades, research has revealed a far broader picture of the events. Membrane contact sites (MCSs) have been recognized as increasingly important actors in cell differentiation, plasticity and maintenance, and, upon dysfunction, responsible for pathological conditions such as cancer and neurodegenerative diseases. Present in multiple organelles and cell types, MCSs promote transport of lipids and Ca2+ homoeostasis, with a range of associated protein families. Interestingly, each MCS displays a unique molecular signature, adapted to organelle functions. This review will explore the literature describing the molecular components and interactions taking place at ER-PM contact sites, their functions, and implications in eukaryotic cells, particularly neurons, with emphasis on lipid transfer proteins and emerging function of SNAREs.
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Affiliation(s)
- Bailey Hewlett
- INSERM U1266, Institut de Psychiatrie et Neurosciences de Paris, Université de Paris, Paris, France
| | - Neha Pratap Singh
- INSERM U1266, Institut de Psychiatrie et Neurosciences de Paris, Université de Paris, Paris, France
| | - Christian Vannier
- INSERM U1266, Institut de Psychiatrie et Neurosciences de Paris, Université de Paris, Paris, France
| | - Thierry Galli
- INSERM U1266, Institut de Psychiatrie et Neurosciences de Paris, Université de Paris, Paris, France.,GHU PARIS Psychiatrie and Neurosciences, Paris, France
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7
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SNAP-25 Puts SNAREs at Center Stage in Metabolic Disease. Neuroscience 2019; 420:86-96. [DOI: 10.1016/j.neuroscience.2018.07.035] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Revised: 07/12/2018] [Accepted: 07/19/2018] [Indexed: 12/20/2022]
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Johnson B, Leek AN, Tamkun MM. Kv2 channels create endoplasmic reticulum / plasma membrane junctions: a brief history of Kv2 channel subcellular localization. Channels (Austin) 2019; 13:88-101. [PMID: 30712450 PMCID: PMC6380216 DOI: 10.1080/19336950.2019.1568824] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The potassium channels Kv2.1 and Kv2.2 are widely expressed throughout the mammalian brain. Kv2.1 provides the majority of delayed rectifying current in rat hippocampus while both channels are differentially expressed in cortex. Particularly unusual is their neuronal surface localization pattern: while half the channel population is freely-diffusive on the plasma membrane as expected from the generalized Singer & Nicolson fluid mosaic model, the other half localizes into micron-sized clusters on the soma, dendrites, and axon initial segment. These clusters contain hundreds of channels, which for Kv2.1, are largely non-conducting. Competing theories of the mechanism underlying Kv2.1 clustering have included static tethering to being corralled by an actin fence. Now, recent work has demonstrated channel clustering is due to formation of endoplasmic reticulum/plasma membrane (ER/PM) junctions through interaction with ER-resident VAMP-associated proteins (VAPs). Interaction between surface Kv2 channels and ER VAPs groups channels together in clusters. ER/PM junctions play important roles in inter-organelle communication: they regulate ion flux, are involved in lipid transfer, and are sites of endo- and exocytosis. Kv2-induced ER/PM junctions are regulated through phosphorylation of the channel C-terminus which in turn regulates VAP binding, providing a rapid means to create or dismantle these microdomains. In addition, insults such as hypoxia or ischemia disrupt this interaction resulting in ER/PM junction disassembly. Kv2 channels are the only known plasma membrane protein to form regulated, injury sensitive junctions in this manner. Furthermore, it is likely that concentrated VAPs at these microdomains sequester additional interactors whose functions are not yet fully understood.
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Affiliation(s)
- Ben Johnson
- a Molecular, Cellular and Integrative Neurosciences Graduate Program , Colorado State University , Fort Collins , CO , USA.,b Department of Biomedical Sciences , Colorado State University , Fort Collins , CO , USA
| | - Ashley N Leek
- a Molecular, Cellular and Integrative Neurosciences Graduate Program , Colorado State University , Fort Collins , CO , USA.,b Department of Biomedical Sciences , Colorado State University , Fort Collins , CO , USA
| | - Michael M Tamkun
- a Molecular, Cellular and Integrative Neurosciences Graduate Program , Colorado State University , Fort Collins , CO , USA.,b Department of Biomedical Sciences , Colorado State University , Fort Collins , CO , USA.,c Department of Biochemistry and Molecular Biology , Colorado State University , Fort Collins , CO , USA
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9
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Jacobson DA, Shyng SL. Ion Channels of the Islets in Type 2 Diabetes. J Mol Biol 2019; 432:1326-1346. [PMID: 31473158 DOI: 10.1016/j.jmb.2019.08.014] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 08/19/2019] [Accepted: 08/21/2019] [Indexed: 02/06/2023]
Abstract
Ca2+ is an essential signal for pancreatic β-cell function. Ca2+ plays critical roles in numerous β-cell pathways such as insulin secretion, transcription, metabolism, endoplasmic reticulum function, and the stress response. Therefore, β-cell Ca2+ handling is tightly controlled. At the plasma membrane, Ca2+ entry primarily occurs through voltage-dependent Ca2+ channels. Voltage-dependent Ca2+ channel activity is dependent on orchestrated fluctuations in the plasma membrane potential or voltage, which are mediated via the activity of many ion channels. During the pathogenesis of type 2 diabetes the β-cell is exposed to stressful conditions, which result in alterations of Ca2+ handling. Some of the changes in β-cell Ca2+ handling that occur under stress result from perturbations in ion channel activity, expression or localization. Defective Ca2+ signaling in the diabetic β-cell alters function, limits insulin secretion and exacerbates hyperglycemia. In this review, we focus on the β-cell ion channels that control Ca2+ handling and how they impact β-cell dysfunction in type 2 diabetes.
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Affiliation(s)
- David A Jacobson
- Department of Molecular Physiology and Biophysics, Vanderbilt University, 7415 MRB4 (Langford), 2213 Garland Avenue, Nashville, TN 37232, USA.
| | - Show-Ling Shyng
- Department of Biochemistry and Molecular Biology, Oregon Health & Science University, L224, MRB 624, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
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Yeh CY, Ye Z, Moutal A, Gaur S, Henton AM, Kouvaros S, Saloman JL, Hartnett-Scott KA, Tzounopoulos T, Khanna R, Aizenman E, Camacho CJ. Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant. Proc Natl Acad Sci U S A 2019; 116:15696-15705. [PMID: 31308225 PMCID: PMC6681760 DOI: 10.1073/pnas.1903401116] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.
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Affiliation(s)
- Chung-Yang Yeh
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Zhaofeng Ye
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- School of Medicine, Tsinghua University, Beijing 100871, China
| | - Aubin Moutal
- Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724
| | - Shivani Gaur
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Amanda M Henton
- Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Stylianos Kouvaros
- Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Jami L Saloman
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Karen A Hartnett-Scott
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Thanos Tzounopoulos
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Rajesh Khanna
- Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724
| | - Elias Aizenman
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
- Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
- Pittsburgh Hearing Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Carlos J Camacho
- Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
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11
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Guerini FR, Ripamonti E, Costa AS, Zanzottera M, Agliardi C, Bolognesi E, Clerici M, Racca V. The Syntaxin-1A gene single nucleotide polymorphism rs4717806 associates with the risk of ischemic heart disease. Medicine (Baltimore) 2019; 98:e15846. [PMID: 31192914 PMCID: PMC6587621 DOI: 10.1097/md.0000000000015846] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Ischemic heart disease (IHD) has a genetic predisposition and a number of cardiovascular risk factors are known to be affected by genetic factors. Development of metabolic syndrome and insulin resistance, strongly influenced by lifestyle and environmental factors, frequently occur in subjects with a genetic susceptibility. The definition of genetic factors influencing disease susceptibility would allow to identify individuals at higher risk and thus needing to be closely monitored.To this end, we focused on a complex of soluble-N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), playing an important role in metabolic syndrome and insulin resistance, involved in endothelial dysfunction and heart disease. We assessed if genetic variants of the SNARE genes are associated with IHD.SNAP25 rs363050, Stx-1A rs4717806, rs2293489, and VAMP2 26bp ins/del genetic polymorphisms were analyzed in a cohort of 100 participants who underwent heart surgery; 56 of them were affected by IHD, while 44 were not. A statistical association of plasma glycemia and insulin resistance, calculated as Triglyceride glucose (TyG) index, was observed in IHD (P < .001 and P = .03, respectively) after binomial logistic stepwise regression analysis, adjusted by age, gender, diabetes positivity, waist circumference, and cholesterol plasma level. Among genetic polymorphisms, rs4717806(A) and rs2293489(T), as well as the rs4717806 - rs2293489 (A-T) haplotype were associated with higher risk for IHD (Pc = .02; Pc = .02; P = .04, respectively). Finally, a statistical association of rs4717806(AA) genotype with higher TyG index in IHD patients (P = .03) was highlighted by multiple regression analysis considering log-transformed biochemical parameters as dependent variable and presence of coronary artery disease, age, gender, waist circumference, presence of diabetes as predictors. These results point to a role of the Stx-1A rs4717806 SNP in IHD, possibly due to its influence on Stx-1A expression and, as a consequence, on insulin secretion and glucose metabolism.
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Affiliation(s)
| | | | | | | | | | | | - Mario Clerici
- IRCCS Fondazione Don Carlo Gnocchi, Milano
- Pathophysiology and Transplantation, University of Milano, Milano, Italy
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12
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Fu J, Githaka JM, Dai X, Plummer G, Suzuki K, Spigelman AF, Bautista A, Kim R, Greitzer-Antes D, Fox JEM, Gaisano HY, MacDonald PE. A glucose-dependent spatial patterning of exocytosis in human β-cells is disrupted in type 2 diabetes. JCI Insight 2019; 5:127896. [PMID: 31085831 DOI: 10.1172/jci.insight.127896] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and an impaired glucose-dependent facilitation of insulin exocytosis. We show in β-cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion, visualized by total internal reflection fluorescence (TIRF) microscopy in 24 of these donors, demonstrate that these are non-random across the surface of β-cells from donors with no diabetes (ND). The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose-dependent and notably impaired in T2D β-cells. Mechanistically, multi-channel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion 'hotspots', while SUMOylation sites at the channel N- (K145) and C-terminus (K470) determine the relative proportion of fusion events occurring within these regions. Thus, a glucose-dependent compartmentalization of fusion, regulated in part by a structural role for Kv2.1, is disrupted in β-cells from donors with type 2 diabetes.
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Affiliation(s)
- Jianyang Fu
- Alberta Diabetes Institute and Department of Pharmacology and
| | | | - Xiaoqing Dai
- Alberta Diabetes Institute and Department of Pharmacology and
| | - Gregory Plummer
- Alberta Diabetes Institute and Department of Pharmacology and
| | - Kunimasa Suzuki
- Alberta Diabetes Institute and Department of Pharmacology and
| | | | - Austin Bautista
- Alberta Diabetes Institute and Department of Pharmacology and
| | - Ryekjang Kim
- Alberta Diabetes Institute and Department of Pharmacology and
| | - Dafna Greitzer-Antes
- Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada
| | | | - Herbert Y Gaisano
- Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada
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13
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Delgado-Ramírez M, Rodríguez-Menchaca AA. Cytoskeleton disruption affects Kv2.1 channel function and its modulation by PIP 2. J Physiol Sci 2019; 69:513-521. [PMID: 30900190 PMCID: PMC10717730 DOI: 10.1007/s12576-019-00671-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Revised: 02/07/2019] [Accepted: 03/07/2019] [Indexed: 11/29/2022]
Abstract
Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15 mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.
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Affiliation(s)
- Mayra Delgado-Ramírez
- Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Venustiano Carranza #2405, Col. Los Filtros, 78210, San Luis Potosí, SLP, Mexico
| | - Aldo A Rodríguez-Menchaca
- Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Venustiano Carranza #2405, Col. Los Filtros, 78210, San Luis Potosí, SLP, Mexico.
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14
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Greitzer-Antes D, Xie L, Qin T, Xie H, Zhu D, Dolai S, Liang T, Kang F, Hardy AB, He Y, Kang Y, Gaisano HY. K v2.1 clusters on β-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3. J Biol Chem 2018; 293:6893-6904. [PMID: 29549124 DOI: 10.1074/jbc.ra118.002703] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2018] [Revised: 03/09/2018] [Indexed: 01/22/2023] Open
Abstract
The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic β cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the β-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.
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Affiliation(s)
- Dafna Greitzer-Antes
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Li Xie
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Tairan Qin
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Huanli Xie
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Dan Zhu
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Subhankar Dolai
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Tao Liang
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Fei Kang
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Alexandre B Hardy
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Yan He
- the Department of Epidemiology and Health Statistics, School of Public Health and Family Medicine, Capital Medical University, Beijing 100050, China
| | - Youhou Kang
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
| | - Herbert Y Gaisano
- From the Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada and
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15
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Daraio T, Valladolid-Acebes I, Brismar K, Bark C. SNAP-25a and SNAP-25b differently mediate interactions with Munc18-1 and Gβγ subunits. Neurosci Lett 2018; 674:75-80. [PMID: 29548989 DOI: 10.1016/j.neulet.2018.03.024] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2017] [Revised: 03/09/2018] [Accepted: 03/12/2018] [Indexed: 02/06/2023]
Abstract
SNAP-25 is a protein involved in regulated membrane fusion and part of the SNARE complex. It exists as two splicing variants, SNAP-25a and SNAP-25b, which differ in 9 out of 206 amino acids. SNAP-25 together with Syntaxin 1 and VAMP-2 forms the ternary SNARE complex essential for mediating activity-dependent release of hormones and neurotransmitters. The functional difference between SNAP-25a and SNAP-25b is poorly understood as both can participate in SNARE complexes and mediate membrane fusion. However, we recently demonstrated that SNAP-25b-deficiency results in metabolic disease and increased insulin secretion. Here we investigated if SNAP-25a and SNAP-25b differently affect interactions with other SNAREs and SNARE-interacting proteins in mouse hippocampus. Adult mice almost exclusively express the SNAP-25b protein in hippocampus whereas SNAP-25b-deficient mice only express SNAP-25a. Immunoprecipitation studies showed no significant differences in amount of Syntaxin 1 and VAMP-2 co-precipitated with the different SNAP-25 isoforms. In contrast, Munc18-1, that preferentially interacts with SNAP-25 via Syntaxin 1 and/or the trimeric SNARE complex, demonstrated an increased ability to bind protein-complexes containing SNAP-25b. Moreover, we found that both SNAP-25 isoforms co-precipitated the Gβγ subunits of the heterotrimeric G proteins, an interaction known to play a role in presynaptic inhibition. We have identified Gβ1 and Gβ2 as the interacting partners of both SNAP-25 isoforms in mouse hippocampus, but Gβ2 was less efficiently captured by SNAP-25a. These results implicate that the two SNAP-25 isoforms could differently mediate protein interactions outside the ternary SNARE core complex and thereby contribute to modulate neurotransmission.
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Affiliation(s)
- Teresa Daraio
- Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden
| | - Ismael Valladolid-Acebes
- Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden
| | - Kerstin Brismar
- Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden
| | - Christina Bark
- Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
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16
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Rorsman P, Ashcroft FM. Pancreatic β-Cell Electrical Activity and Insulin Secretion: Of Mice and Men. Physiol Rev 2018; 98:117-214. [PMID: 29212789 PMCID: PMC5866358 DOI: 10.1152/physrev.00008.2017] [Citation(s) in RCA: 518] [Impact Index Per Article: 74.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Revised: 05/30/2017] [Accepted: 06/18/2017] [Indexed: 12/14/2022] Open
Abstract
The pancreatic β-cell plays a key role in glucose homeostasis by secreting insulin, the only hormone capable of lowering the blood glucose concentration. Impaired insulin secretion results in the chronic hyperglycemia that characterizes type 2 diabetes (T2DM), which currently afflicts >450 million people worldwide. The healthy β-cell acts as a glucose sensor matching its output to the circulating glucose concentration. It does so via metabolically induced changes in electrical activity, which culminate in an increase in the cytoplasmic Ca2+ concentration and initiation of Ca2+-dependent exocytosis of insulin-containing secretory granules. Here, we review recent advances in our understanding of the β-cell transcriptome, electrical activity, and insulin exocytosis. We highlight salient differences between mouse and human β-cells, provide models of how the different ion channels contribute to their electrical activity and insulin secretion, and conclude by discussing how these processes become perturbed in T2DM.
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Affiliation(s)
- Patrik Rorsman
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, United Kingdom; Department of Neuroscience and Physiology, Metabolic Research Unit, Göteborg, Sweden; and Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | - Frances M Ashcroft
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, United Kingdom; Department of Neuroscience and Physiology, Metabolic Research Unit, Göteborg, Sweden; and Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
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17
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Daraio T, Bombek LK, Gosak M, Valladolid-Acebes I, Klemen MS, Refai E, Berggren PO, Brismar K, Rupnik MS, Bark C. SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of Ca 2+oscillations in β cell networks. Sci Rep 2017; 7:7744. [PMID: 28798351 PMCID: PMC5552776 DOI: 10.1038/s41598-017-08082-y] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2017] [Accepted: 07/04/2017] [Indexed: 01/02/2023] Open
Abstract
SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic β cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic β cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged Ca2+ in β cells within pancreatic slices showed no significant differences in Ca2+-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if Ca2+ handling was affected in glucose-stimulated β cells using intracellular Ca2+-imaging and found premature activation and delayed termination of [Ca2+]i elevations. These findings were accompanied by less synchronized Ca2+-oscillations and hence more segregated functional β cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic β cells, except for participating in the core SNARE complex, is necessary for accurate regulation of Ca2+-dynamics.
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Affiliation(s)
- Teresa Daraio
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden
| | - Lidija Križančić Bombek
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000, Maribor, Slovenia
| | - Marko Gosak
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000, Maribor, Slovenia.,Department of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, SI-2000, Maribor, Slovenia
| | - Ismael Valladolid-Acebes
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden
| | - Maša Skelin Klemen
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000, Maribor, Slovenia
| | - Essam Refai
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden
| | - Per-Olof Berggren
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden
| | - Kerstin Brismar
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden
| | - Marjan Slak Rupnik
- Institute of Physiology, Faculty of Medicine, University of Maribor, SI-2000, Maribor, Slovenia. .,Center for Physiology and Pharmacology, Medical University of Vienna, A-1090, Vienna, Austria.
| | - Christina Bark
- The Rolf Luft Research Center for Diabetes and Endocrinology, Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76, Stockholm, Sweden.
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18
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Lee SM, Baik J, Nguyen D, Nguyen V, Liu S, Hu Z, Abbott GW. Kcne2 deletion impairs insulin secretion and causes type 2 diabetes mellitus. FASEB J 2017; 31:2674-2685. [PMID: 28280005 DOI: 10.1096/fj.201601347] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Accepted: 02/21/2017] [Indexed: 02/05/2023]
Abstract
Type 2 diabetes mellitus (T2DM) represents a rapidly increasing threat to global public health. T2DM arises largely from obesity, poor diet, and lack of exercise, but it also involves genetic predisposition. Here we report that the KCNE2 potassium channel transmembrane regulatory subunit is expressed in human and mouse pancreatic β cells. Kcne2 deletion in mice impaired glucose tolerance as early as 5 wk of age in pups fed a Western diet, ultimately causing diabetes. In adult mice fed normal chow, skeletal muscle expression of insulin receptor β and insulin receptor substrate 1 were down-regulated 2-fold by Kcne2 deletion, characteristic of T2DM. Kcne2 deletion also caused extensive pancreatic transcriptome changes consistent with facets of T2DM, including endoplasmic reticulum stress, inflammation, and hyperproliferation. Kcne2 deletion impaired β-cell insulin secretion in vitro up to 8-fold and diminished β-cell peak outward K+ current at positive membrane potentials, but also left-shifted its voltage dependence and slowed inactivation. Interestingly, we also observed an aging-dependent reduction in β-cell outward currents in both Kcne2+/+ and Kcne2-/- mice. Our results demonstrate that KCNE2 is required for normal β-cell electrical activity and insulin secretion, and that Kcne2 deletion causes T2DM. KCNE2 may regulate multiple K+ channels in β cells, including the T2DM-linked KCNQ1 potassium channel α subunit.-Lee, S. M., Baik, J., Nguyen, D., Nguyen, V., Liu, S., Hu, Z., Abbott, G. W. Kcne2 deletion impairs insulin secretion and causes type 2 diabetes mellitus.
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Affiliation(s)
- Soo Min Lee
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA
| | - Jasmine Baik
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA
| | - Dara Nguyen
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA
| | - Victoria Nguyen
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA
| | - Shiwei Liu
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA
| | - Zhaoyang Hu
- Laboratory of Anesthesiology and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Geoffrey W Abbott
- Bioelectricity Laboratory, Department of Pharmacology and Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA;
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19
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Al-Daghri NM, Costa AS, Alokail MS, Zanzottera M, Alenad AM, Mohammed AK, Clerici M, Guerini FR. Synaptosomal Protein of 25 kDa (Snap25) Polymorphisms Associated with Glycemic Parameters in Type 2 Diabetes Patients. J Diabetes Res 2016; 2016:8943092. [PMID: 26779543 PMCID: PMC4686705 DOI: 10.1155/2016/8943092] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Revised: 07/24/2015] [Accepted: 07/28/2015] [Indexed: 11/22/2022] Open
Abstract
A possible role of Snap25 polymorphisms in type 2 diabetes mellitus (T2DM) was evaluated by analyzing three SNPs within intron 1 in a region known to affect the gene expression in vitro. Genomic DNA from 1019 Saudi individuals (489 confirmed T2DM and 530 controls) was genotyped for SNPs rs363039, rs363043, and rs363050 in Snap25 using the TaqMan Genotyping Assay. Significantly higher levels of fasting glucose and HbA1c were detected in T2DM patients carrying the rs363050 (AG/GG) genotypes compared to the (AA) genotype (f = 4.41, df = 1, and p = 0.03 and f = 5.31, df = 1, and p = 0.03, resp.). In these same patients, insulin levels were significantly decreased compared to the (AA) individuals (f = 7.29, df = 1, and p = 0.009). Significant associations were detected between rs363050 (AG/GG) genotypes and increasing fasting glucose levels (p = 0.01 and OR: 1.05), HbA1c levels (OR: 5.06 and p = 0.02), and lower insulinemia (p = 0.03 and OR: 0.95) in T2DM patients. The minor Snap25 rs363050 (G) allele, which results in a reduced expression of Snap25, is associated with altered glycemic parameters in T2DM possibly because of reduced functionality in the exocytotic machinery leading to suboptimal release of insulin.
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Affiliation(s)
- Nasser M. Al-Daghri
- Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
- Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
- *Nasser M. Al-Daghri:
| | | | - Majed S. Alokail
- Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
- Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | | | - Amal M. Alenad
- School of Biological Sciences, University of Southampton, Southampton SO17 1 BJ, UK
| | - Abdul Khader Mohammed
- Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
- Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Mario Clerici
- Fondazione Don C Gnocchi, IRCCS, 20148 Milano, Italy
- Università degli Studi di Milano, 20122 Milano, Italy
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20
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Zhang Y, Wang H, Guo Q, Li X, Gao J, Liu Y, Yang C, Niu L, Yang J. PI3K is involved in P2Y receptor-regulated cAMP /Epac/Kv channel signaling pathway in pancreatic β cells. Biochem Biophys Res Commun 2015; 465:714-8. [PMID: 26296468 DOI: 10.1016/j.bbrc.2015.08.057] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2015] [Accepted: 08/13/2015] [Indexed: 12/20/2022]
Abstract
P2Y receptors (P2YR) are a family of purinergic G protein-coupled receptors, which could be stimulated by extracellular nucleotides. In pancreatic β cells, activation of P2YR has long been shown to stimulate insulin secretion in a glucose-dependent manner. Previously, we reported that P2YR-modulated insulin secretion is mediated by a cAMP/Epac/Kv channel pathway. However, the interaction between Epac and the Kv channel in P2YR-modulated insulin secretion remains unclear. In this study, we used patch-clamp technique and insulin secretion assay to investigate the potential molecules that may link Epac to Kv channel inhibition induced by P2YR activation. We identified that phosphatidylinositide 3-kinase, which mediates P2YR-regulated insulin secretion, is a critical mediator between Epac and the Kv channel.
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Affiliation(s)
- Yi Zhang
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Hui Wang
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Qing Guo
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Xiaodong Li
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Jingying Gao
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Yunfeng Liu
- Department of Endocrinology, The First Hospital of Shanxi Medical University, Shanxi Medical University, Taiyuan, China.
| | - Caihong Yang
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Longgang Niu
- Department of Pharmacology, Shanxi Medical University, Taiyuan, China.
| | - Jing Yang
- Department of Endocrinology, The First Hospital of Shanxi Medical University, Shanxi Medical University, Taiyuan, China.
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21
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Chen G, Hu T, Li Q, Li J, Jia Y, Wang Z. Expression of synaptosomal-associated protein-25 in the rat brain after subarachnoid hemorrhage. Neural Regen Res 2014; 8:2693-702. [PMID: 25206580 PMCID: PMC4145993 DOI: 10.3969/j.issn.1673-5374.2013.29.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Accepted: 08/09/2013] [Indexed: 11/18/2022] Open
Abstract
Synaptosomal-associated protein-25 is an important factor for synaptic functions and cognition. In this study, subarachnoid hemorrhage models with spatial learning disorder were established through a blood injection into the chiasmatic cistern. Immunohistochemical staining and western blot analysis results showed that synaptosomal-associated protein-25 expression in the temporal lobe, hippocampus, and cerebellum significantly lower at days 1 and 3 following subarachnoid morrhage. Our findings indicate that synaptosomal-associated protein-25 expression was down-regulated in the rat brain after subarachnoid hemorrhage.
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Affiliation(s)
- Gang Chen
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
| | - Tong Hu
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China ; Department of Neurosurgery, Yixing People's Hospital, Yixing 214200, Jiangsu Province, China
| | - Qi Li
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
| | - Jianke Li
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
| | - Yang Jia
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
| | - Zhong Wang
- Department of Neurosurgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
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22
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Caceres PS, Mendez M, Ortiz PA. Vesicle-associated membrane protein 2 (VAMP2) but Not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl- co-transporter NKCC2 in thick ascending limbs. J Biol Chem 2014; 289:23951-62. [PMID: 25008321 PMCID: PMC4156046 DOI: 10.1074/jbc.m114.589333] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.
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Affiliation(s)
- Paulo S Caceres
- From the Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202 and the Department of Physiology, Wayne State University, Detroit, Michigan 48202
| | - Mariela Mendez
- From the Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202 and
| | - Pablo A Ortiz
- From the Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202 and the Department of Physiology, Wayne State University, Detroit, Michigan 48202
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23
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Fridlyand LE, Jacobson DA, Philipson LH. Ion channels and regulation of insulin secretion in human β-cells: a computational systems analysis. Islets 2013; 5:1-15. [PMID: 23624892 PMCID: PMC3662377 DOI: 10.4161/isl.24166] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
In mammals an increase in glucose leads to block of ATP dependent potassium channels in pancreatic β cells leading to membrane depolarization. This leads to the repetitive firing of action potentials that increases calcium influx and triggers insulin granule exocytosis. Several important differences between species in this process suggest that a dedicated human-oriented approach is advantageous as extrapolating from rodent data may be misleading in several respects. We examined depolarization-induced spike activity in pancreatic human islet-attached β-cells employing whole-cell patch-clamp methods. We also reviewed the literature concerning regulation of insulin secretion by channel activity and constructed a data-based computer model of human β cell function. The model couples the Hodgkin-Huxley-type ionic equations to the equations describing intracellular Ca²⁺ homeostasis and insulin release. On the basis of this model we employed computational simulations to better understand the behavior of action potentials, calcium handling and insulin secretion in human β cells under a wide range of experimental conditions. This computational system approach provides a framework to analyze the mechanisms of human β cell insulin secretion.
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24
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Deutsch E, Weigel AV, Akin EJ, Fox P, Hansen G, Haberkorn CJ, Loftus R, Krapf D, Tamkun MM. Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane. Mol Biol Cell 2012; 23:2917-29. [PMID: 22648171 PMCID: PMC3408418 DOI: 10.1091/mbc.e12-01-0047] [Citation(s) in RCA: 81] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Voltage-gated K+ (Kv) channels regulate membrane potential in many cell types. Although the channel surface density and location must be well controlled, little is known about Kv channel delivery and retrieval on the cell surface. The Kv2.1 channel localizes to micron-sized clusters in neurons and transfected human embryonic kidney (HEK) cells, where it is nonconducting. Because Kv2.1 is postulated to be involved in soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion, we examined the hypothesis that these surface clusters are specialized platforms involved in membrane protein trafficking. Total internal reflection–based fluorescence recovery after photobleaching studies and quantum dot imaging of single Kv2.1 channels revealed that Kv2.1-containing vesicles deliver cargo at the Kv2.1 surface clusters in both transfected HEK cells and hippocampal neurons. More than 85% of cytoplasmic and recycling Kv2.1 channels was delivered to the cell surface at the cluster perimeter in both cell types. At least 85% of recycling Kv1.4, which, unlike Kv2.1, has a homogeneous surface distribution, is also delivered here. Actin depolymerization resulted in Kv2.1 exocytosis at cluster-free surface membrane. These results indicate that one nonconducting function of Kv2.1 is to form microdomains involved in membrane protein trafficking. This study is the first to identify stable cell surface platforms involved in ion channel trafficking.
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Affiliation(s)
- Emily Deutsch
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Aubrey V. Weigel
- School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523
| | - Elizabeth J. Akin
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Phil Fox
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Gentry Hansen
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | | | - Rob Loftus
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Diego Krapf
- School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523
- Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, CO 80523
| | - Michael M. Tamkun
- Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523
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25
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Balse E, Steele DF, Abriel H, Coulombe A, Fedida D, Hatem SN. Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes. Physiol Rev 2012; 92:1317-58. [DOI: 10.1152/physrev.00041.2011] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.
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Affiliation(s)
- Elise Balse
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
| | - David F. Steele
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
| | - Hugues Abriel
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
| | - Alain Coulombe
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
| | - David Fedida
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
| | - Stéphane N. Hatem
- Institute of Cardiometabolism and Nutrition, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Heart and Metabolism Division, Paris, France; Institut National de la Santé et de la Recherche Médicale UMR_S956, Paris, France; Université Pierre et Marie Curie, Paris, France; Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada; and Department of Clinical Research University of Bern, Bern, Switzerland
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26
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Dai XQ, Manning Fox JE, Chikvashvili D, Casimir M, Plummer G, Hajmrle C, Spigelman AF, Kin T, Singer-Lahat D, Kang Y, Shapiro AMJ, Gaisano HY, Lotan I, Macdonald PE. The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function. Diabetologia 2012; 55:1709-1720. [PMID: 22411134 DOI: 10.1007/s00125-012-2512-6] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2011] [Accepted: 01/24/2012] [Indexed: 02/07/2023]
Abstract
AIMS/HYPOTHESIS It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.
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Affiliation(s)
- X Q Dai
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada T6G 2E1
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27
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Hao S, Bao YM, An LJ, Cheng W, Zhao RG, Bi J, Wang HS, Sun CS, Liu JW, Jiang B. Effects of Resibufogenin and Cinobufagin on voltage-gated potassium channels in primary cultures of rat hippocampal neurons. Toxicol In Vitro 2011; 25:1644-53. [DOI: 10.1016/j.tiv.2011.07.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Revised: 05/29/2011] [Accepted: 07/04/2011] [Indexed: 10/17/2022]
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28
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MacDonald PE. Signal integration at the level of ion channel and exocytotic function in pancreatic β-cells. Am J Physiol Endocrinol Metab 2011; 301:E1065-9. [PMID: 21934040 DOI: 10.1152/ajpendo.00426.2011] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Whole body energy balance is ensured by the exquisite control of insulin secretion, the dysregulation of which has serious consequences. Although a great deal has been learned about the control of insulin secretion from pancreatic β-cells in the past 30 years, there remains much to be understood about the molecular mechanisms and interactions that underlie the precise control of this process. Numerous molecular interactions at the plasma membrane mediate the excitatory and amplifying events involved in insulin secretion; this includes interactions between ion channels, signal transduction machinery, and exocytotic proteins. The present Perspectives article considers evidence that key membrane and membrane-associated proteins essential to insulin secretion are regulated in concert as a functional unit, ensuring an integrated excitatory and exocytotic response to the signals that control insulin secretion.
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Affiliation(s)
- Patrick E MacDonald
- Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.
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29
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Chao CC, Mihic A, Tsushima RG, Gaisano HY. SNARE protein regulation of cardiac potassium channels and atrial natriuretic factor secretion. J Mol Cell Cardiol 2011; 50:401-7. [DOI: 10.1016/j.yjmcc.2010.11.018] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2010] [Revised: 11/17/2010] [Accepted: 11/19/2010] [Indexed: 01/28/2023]
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30
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Yao H, Zhou K, Yan D, Li M, Wang Y. The Kv2.1 channels mediate neuronal apoptosis induced by excitotoxicity. J Neurochem 2010. [DOI: 10.1111/j.0022-3042.2008.05834.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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31
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Vikman J, Svensson H, Huang YC, Kang Y, Andersson SA, Gaisano HY, Eliasson L. Truncation of SNAP-25 reduces the stimulatory action of cAMP on rapid exocytosis in insulin-secreting cells. Am J Physiol Endocrinol Metab 2009; 297:E452-61. [PMID: 19509185 DOI: 10.1152/ajpendo.90585.2008] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Synaptosomal protein of 25 kDa (SNAP-25) is important for Ca(2+)-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis. In insulin-secreting INS-1 cells overexpressing wild-type SNAP-25 (SNAP-25(WT)), rapid exocytosis was stimulated more than threefold by cAMP, similar to the situation in nontransfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 (SNAP-25(1-197)) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in the presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the readily releasable pool, especially from the immediately releasable pool, whereas cAMP enhanced mobilization of granules from the reserve pool in both SNAP-25(1-197) (P < 0.01) and SNAP-25(WT) (P < 0.05) cells. This was supported by hormone release measurements. Augmentation of the immediately releasable pool by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin-secreting cells.
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Affiliation(s)
- Jenny Vikman
- Department of Clinical Sciences Lund, Biomedical Center, Lund University Diabetes Centre, Lund, Sweden
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32
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Redman PT, Hartnett KA, Aras MA, Levitan ES, Aizenman E. Regulation of apoptotic potassium currents by coordinated zinc-dependent signalling. J Physiol 2009; 587:4393-404. [PMID: 19622611 DOI: 10.1113/jphysiol.2009.176321] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Oxidant-liberated intracellular Zn(2+) regulates neuronal apoptosis via an exocytotic membrane insertion of Kv2.1-encoded ion channels, resulting in an enhancement of voltage-gated K(+) currents and a loss of intracellular K(+) that is necessary for caspase-mediated proteolysis. In the present study we show that an N-terminal tyrosine of Kv2.1 (Y124), which is a known target of Src kinase, is critical for the apoptotic current surge. Moreover, we demonstrate that Y124 works in concert with a C-terminal serine (S800) target of p38 mitogen-activated protein kinase (MAPK) to regulate Kv2.1-mediated current enhancement. While Zn(2+) was previously shown to activate p38, we show here that this metal inhibits cytoplasmic protein tyrosine phosphatase (Cyt-PTPepsilon), which specifically targets Y124. Importantly, a point mutation of Y124 to a non-phosphorylatable residue or over-expression of Cyt-PTPepsilon protects cells from injury. Kv2.1-encoded channels thus regulate neuronal survival by providing a converging input for two Zn(2+)-dependent signal transduction cascades.
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Affiliation(s)
- Patrick T Redman
- Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
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33
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Feinshreiber L, Singer-Lahat D, Ashery U, Lotan I. Voltage-gated potassium channel as a facilitator of exocytosis. Ann N Y Acad Sci 2009; 1152:87-92. [PMID: 19161379 DOI: 10.1111/j.1749-6632.2008.03997.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Voltage-gated ion channels are well characterized for their function in excitability signals. Accumulating studies, however, have established an ion-independent function for the major classes of ion channels in cellular signaling. During the last few years we established a novel role for Kv2.1, a voltage-gated potassium (Kv) channel, classically known for its role of repolarizing the membrane potential, in facilitation of exocytosis. Kv2.1 induces facilitation of depolarization-induced release through its direct interaction with syntaxin, a protein component of the exocytotic machinery, independently of the potassium ion flow through the channel's pore. Here, we review our recent studies, further characterize the phenomena (using chromaffin cells and carbon fiber amperometry), and suggest plausible mechanisms that can underlie this facilitation of release.
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Affiliation(s)
- Lori Feinshreiber
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel
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34
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SNAP-25(1-180) enhances insulin secretion by blocking Kv2.1 channels in rat pancreatic islet beta-cells. Biochem Biophys Res Commun 2008; 379:812-6. [PMID: 19103161 DOI: 10.1016/j.bbrc.2008.12.059] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2008] [Accepted: 12/11/2008] [Indexed: 11/21/2022]
Abstract
Voltage-gated outward K(+) currents from pancreatic islet beta-cells are known to repolarize the action potential during a glucose stimulus, and consequently to modulate Ca(2+) entry and insulin secretion. The voltage gated K(+) (Kv) channel, Kv2.1, which is expressed in rat islet beta-cells, mediates over 60% of the Kv outward K(+) currents. A novel peptidyl inhibitor of Kv2.1/Kv2.2 channels, guangxitoxin (GxTX)-1, has been shown to enhance glucose-stimulated insulin secretion. Here, we show that SNAP-25(1-180) (S180), an N-terminal SNAP-25 domain, but not SNAP-25(1-206) (S206), inhibits Kv current and enhances glucose-dependent insulin secretion from rat pancreatic islet beta-cells, and furthermore, this enhancement was induced by the blockade of the Kv2.1 current. This study indicates that the Kv2.1 channel is a potential target for novel therapeutic agent design for the treatment of type 2 diabetes. This target may possess advantages over currently-used therapies, which modulate insulin secretion in a glucose-independent manner.
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35
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Yao H, Zhou K, Yan D, Li M, Wang Y. The Kv2.1 channels mediate neuronal apoptosis induced by excitotoxicity. J Neurochem 2008; 108:909-19. [PMID: 19077057 DOI: 10.1111/j.1471-4159.2008.05834.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Chronic loss of intracellular K(+) can induce neuronal apoptosis in pathological conditions. However, the mechanism by which the K(+) channels are regulated in this process remains largely unknown. Here, we report that the increased membrane expression of Kv2.1 proteins in cortical neurons deprived of serum, a condition known to induce K(+) loss, promotes neuronal apoptosis. The increase in I(K) current density and apoptosis in the neurons deprived of serum were inhibited by a dominant negative form of Kv2.1 and MK801, an antagonist to NMDA receptors. The membrane level of Kv2.1 and its interaction with SNAP25 were increased, whereas the Kv2.1 phosphorylation was inhibited in the neurons deprived of serum. Botulinum neurotoxin, an agent known to prevent formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, suppressed the increase in I(K) current density. Together, these results suggest that NMDA receptor-dependent Kv2.1 membrane translocation is regulated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor-dependent vesicular trafficking mechanism and is responsible for neuronal cell death induced by chronic loss of K(+).
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Affiliation(s)
- Hailan Yao
- The Graduate School, Chinese Academy of Science, Shanghai, China
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36
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Tsuk S, Lvov A, Michaelevski I, Chikvashvili D, Lotan I. Formation of the full SNARE complex eliminates interactions of its individual protein components with the Kv2.1 channel. Biochemistry 2008; 47:8342-9. [PMID: 18636750 DOI: 10.1021/bi800512p] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.
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Affiliation(s)
- Sharon Tsuk
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel
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37
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Lvov A, Chikvashvili D, Michaelevski I, Lotan I. VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation. Pflugers Arch 2008; 456:1121-36. [PMID: 18542995 DOI: 10.1007/s00424-008-0468-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2007] [Revised: 01/06/2008] [Accepted: 01/22/2008] [Indexed: 11/25/2022]
Abstract
Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.
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Affiliation(s)
- Anatoli Lvov
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, 69978, Ramat-Aviv, Israel
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38
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Affiliation(s)
- James A McNew
- Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street MS-140, Houston, Texas 77251-1892, USA.
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Lovis P, Gattesco S, Regazzi R. Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by microRNAs. Biol Chem 2008; 389:305-12. [DOI: 10.1515/bc.2008.026] [Citation(s) in RCA: 214] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Abstract
Fine-tuning of insulin secretion from pancreatic β-cells participates in blood glucose homeostasis. Defects in this process can lead to chronic hyperglycemia and diabetes mellitus. Several proteins controlling insulin exocytosis have been identified, but the mechanisms regulating their expression remain poorly understood. Here, we show that two non-coding microRNAs, miR124a and miR96, modulate the expression of proteins involved in insulin exocytosis and affect secretion of the β-cell line MIN6B1. miR124a increases the levels of SNAP25, Rab3A and synapsin-1A and decreases those of Rab27A and Noc2. Inhibition of Rab27A expression is mediated by direct binding to the 3′-untranslated region of Rab27A mRNA. The effect on the other genes is indirect and linked to changes in mRNA levels. Over-expression of miR124a leads to exaggerated hormone release under basal conditions and a reduction in glucose-induced secretion. miR96 increases mRNA and protein levels of granuphilin, a negative modulator of insulin exocytosis, and decreases the expression of Noc2, resulting in lower capacity of MIN6B1 cells to respond to secretagogues. Our data identify miR124a and miR96 as novel regulators of the expression of proteins playing a critical role in insulin exocytosis and in the release of other hormones and neurotransmitters.
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Singer-Lahat D, Chikvashvili D, Lotan I. Direct interaction of endogenous Kv channels with syntaxin enhances exocytosis by neuroendocrine cells. PLoS One 2008; 3:e1381. [PMID: 18167541 PMCID: PMC2148073 DOI: 10.1371/journal.pone.0001381] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2007] [Accepted: 11/26/2007] [Indexed: 11/18/2022] Open
Abstract
K+ efflux through voltage-gated K+ (Kv) channels can attenuate the release of neurotransmitters, neuropeptides and hormones by hyperpolarizing the membrane potential and attenuating Ca2+ influx. Notably, direct interaction between Kv2.1 channels overexpressed in PC12 cells and syntaxin has recently been shown to facilitate dense core vesicle (DCV)-mediated release. Here, we focus on endogenous Kv2.1 channels and show that disruption of their interaction with native syntaxin after ATP-dependent priming of the vesicles by Kv2.1 syntaxin–binding peptides inhibits Ca2+ -triggered exocytosis of DCVs from cracked PC12 cells in a specific and dose-dependent manner. The inhibition cannot simply be explained by the impairment of the interaction of syntaxin with its SNARE cognates. Thus, direct association between endogenous Kv2.1 and syntaxin enhances exocytosis and in combination with the Kv2.1 inhibitory effect to hyperpolarize the membrane potential, could contribute to the known activity dependence of DCV release in neuroendocrine cells and in dendrites where Kv2.1 commonly expresses and influences release.
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Affiliation(s)
- Dafna Singer-Lahat
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel
| | - Dodo Chikvashvili
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel
| | - Ilana Lotan
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel
- * To whom correspondence should be addressed. E-mail:
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Botulinum neurotoxin A and neurotoxin E cleavage products of synaptosome-associated protein of 25 kd exhibit distinct actions on pancreatic islet beta-cell Kv2.1 channel gating. Pancreas 2008; 36:10-7. [PMID: 18192874 DOI: 10.1097/mpa.0b013e31812eee28] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVES Synaptosome-associated protein of 25 kd (SNAP-25) regulates pancreatic islet beta-cell-delayed rectifier K channels (Kv2.1) in addition to insulin exocytosis. Botulinum neurotoxin A (BoNT/A) and E (BoNT/E) cleavage and presumed deletion of SNAP-25 have been used to examine SNAP-25 function. We hypothesized that proteolytic products of SNAP-25 (206 amino acids) resulting from BoNT/A and BoNT/E cleavage, SNAP-25(1-197) and SNAP-25(1-180), have independent actions on beta-cell Kv gating. METHODS We examined by confocal microscopy and immunoblotting BoNT/A and BoNT/E cleavage of SNAP-25 to these N-terminal fragments, and the consequent effects of these BoNTs and SNAP-25 fragments on Kv currents in rat beta cells and MIN6 cells by patch clamp electrophysiology. RESULTS Confocal microscopy and immunoblotting showed that MIN6 cells transfected with BoNT/A or BoNT/E generated SNAP-25(1-197) and SNAP-25(1-180) fragments that were retained in the cytosol. Both BoNTs caused increased rate of channel activation and slowed channel inactivation, mimicked by these SNAP-25 fragments, but not full-length SNAP-25. These SNAP-25 fragments potentiated tetraethylammonium block of beta-cell Kv currents. CONCLUSIONS BoNT/A or BoNT/E treatment of beta cells generates N-terminal SNAP-25 fragments that are retained in beta cells to directly influence Kv channel gating in a manner distinct from full-length SNAP-25, contributing to overall actions of these BoNTs on insulin secretion.
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Frolov RV, Berim IG, Singh S. Inhibition of delayed rectifier potassium channels and induction of arrhythmia: a novel effect of celecoxib and the mechanism underlying it. J Biol Chem 2007; 283:1518-1524. [PMID: 17984087 DOI: 10.1074/jbc.m708100200] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Selective inhibitors of cyclooxygenase-2 (COX-2), such as rofecoxib (Vioxx), celecoxib (Celebrex), and valdecoxib (Bextra), have been developed for treating arthritis and other musculoskeletal complaints. Selective inhibition of COX-2 over COX-1 results in preferential decrease in prostacyclin production over thromboxane A2 production, thus leading to less gastric effects than those seen with nonselective COX inhibitors such as acetylsalicylic acid (aspirin). Here we show a novel effect of celecoxib via a mechanism that is independent of COX-2 inhibition. The drug inhibited the delayed rectifier (Kv2) potassium channels from Drosophila, rats, and humans and led to pronounced arrhythmia in Drosophila heart and arrhythmic beating of rat heart cells in culture. These effects occurred despite the genomic absence of cyclooxygenases in Drosophila and the failure of acetylsalicylic acid, a potent inhibitor of both COX-1 and COX-2, to inhibit rat Kv2.1 channels. A genetically null mutant of Drosophila Shab (Kv2) channels reproduced the cardiac effect of celecoxib, and the drug was unable to further enhance the effect of the mutation. These observations reveal an unanticipated effect of celecoxib on Drosophila hearts and on heart cells from rats, implicating the inhibition of Kv2 channels as the mechanism underlying this effect.
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Affiliation(s)
- Roman V Frolov
- Department of Pharmacology and Toxicology, State University of New York, Buffalo, New York 14214
| | - Ilya G Berim
- Department of Medicine, State University of New York, Buffalo, New York 14214
| | - Satpal Singh
- Department of Pharmacology and Toxicology, State University of New York, Buffalo, New York 14214.
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Abstract
Coordinated electrical activity allows pancreatic beta-cells to respond to secretagogues with calcium entry followed by insulin secretion. Metabolism of glucose affects multiple membrane proteins including ion channels, transporters and pumps that collaborate in a cascade of electrical activity resulting in insulin release. Glucose induces beta-cell depolarization resulting in the firing of action potentials (APs), which are the primary electrical signal of the beta-cell. They are shaped by orchestrated activation of ion channels. Here we give an overview of the voltage-gated potassium (Kv) channels of the beta-cell, which are responsible in part for the falling phase of the AP, and how their regulation affects insulin secretion. beta cells contain several Kv channels allowing dynamic integration of multiple signals on repolarization of glucose-stimulated APs. Recent studies on Kv channel regulation by cAMP and arachidonic acid and on the Kv2.1 null mouse have greatly increased our understanding of beta-cell excitation-secretion coupling.
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Affiliation(s)
- D A Jacobson
- Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
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Thurmond DC. Regulation of Insulin Action and Insulin Secretion by SNARE-Mediated Vesicle Exocytosis. MECHANISMS OF INSULIN ACTION 2007:52-70. [DOI: 10.1007/978-0-387-72204-7_3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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45
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Leung YM, Kwan EP, Ng B, Kang Y, Gaisano HY. SNAREing voltage-gated K+ and ATP-sensitive K+ channels: tuning beta-cell excitability with syntaxin-1A and other exocytotic proteins. Endocr Rev 2007; 28:653-63. [PMID: 17878408 DOI: 10.1210/er.2007-0010] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca(2+) channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet beta-cells, ATP-sensitive K(+) (K(ATP)) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca(2+) channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K(+) (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in beta-cells) and the sulfonylurea receptor subunit of beta-cell K(ATP) channel to modify their gating behaviors. A model has proposed that the conformational changes of syntaxin-1A during exocytosis induce distinct functional modulations of K(ATP) and Kv2.1 channels in a manner that optimally regulates cell excitability and insulin secretion. Other proteins involved in exocytosis, such as Munc-13, tomosyn, rab3a-interacting molecule, and guanyl nucleotide exchange factor II, have also been implicated in direct or indirect regulation of beta-cell ion channel activities and excitability. This review discusses this interesting aspect that exocytotic proteins not only promote secretion per se, but also fine-tune beta-cell excitability via modulation of ion channel gating.
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Affiliation(s)
- Yuk M Leung
- Departmnet of Physiology, China Medical University, Taichung 40402, Taiwan.
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Jacobson DA, Kuznetsov A, Lopez JP, Kash S, Ämmälä CE, Philipson LH. Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion. Cell Metab 2007; 6:229-35. [PMID: 17767909 PMCID: PMC2699758 DOI: 10.1016/j.cmet.2007.07.010] [Citation(s) in RCA: 140] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/04/2007] [Revised: 07/10/2007] [Accepted: 07/30/2007] [Indexed: 11/18/2022]
Abstract
Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.
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Affiliation(s)
- David A. Jacobson
- Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA
- Correspondence: (D.A.J.), (L.H.P.)
| | - Andrey Kuznetsov
- Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA
| | - James P. Lopez
- Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA
| | - Shera Kash
- Deltagen Inc., San Mateo, California 94403, USA
| | - Carina E. Ämmälä
- Department of Metabolic Diseases, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA
| | - Louis H. Philipson
- Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA
- Correspondence: (D.A.J.), (L.H.P.)
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Mohapatra DP, Vacher H, Trimmer JS. The Surprising Catch of a Voltage-Gated Potassium Channel in a Neuronal SNARE. ACTA ACUST UNITED AC 2007; 2007:pe37. [PMID: 17609479 DOI: 10.1126/stke.3932007pe37] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Among ion channels, voltage-gated calcium channels have been considered unique in their ability to mediate signaling events independent of the flow of ions through their pore. A voltage-gated potassium channel termed Kv2.1 has been identified as playing a role remarkably similar to one ion-independent function of calcium channels, facilitating regulated exocytosis through a direct interaction with a t-SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] component of the vesicle release machinery. Kv2.1 overexpression enhances depolarization-induced secretion from the neuroendocrine-like PC12 cell line, and a nonconducting Kv2.1 mutant can accomplish the same feat. Kv2.1 interacts directly with syntaxin 1A, a plasma membrane t-SNARE component of the vesicle docking and fusion apparatus. Deletion of the syntaxin 1A-binding segment from Kv2.1 abolishes its ability to promote vesicle release, supporting a mechanism whereby Kv2.1 presumably transfers voltage-dependent conformational changes induced by membrane depolarization to interacting t-SNAREs to affect exocytosis. Kv2.1, a major mediator of electrical events in central neurons, cardiac and smooth muscle, and pancreatic beta cells, must now also be recognized as a physical mediator of secretion. That Kv2.1 is phosphorylated at numerous sites within the syntaxin 1A binding segment raises the possibility that its role in secretion may be dynamically regulated by diverse signaling events.
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Affiliation(s)
- Durga P Mohapatra
- Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616, USA
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Neshatian L, Leung YM, Kang Y, Gao X, Xie H, Tsushima RG, Gaisano HY, Diamant NE. Distinct modulation of Kv1.2 channel gating by wild type, but not open form, of syntaxin-1A. Am J Physiol Gastrointest Liver Physiol 2007; 292:G1233-42. [PMID: 17234891 DOI: 10.1152/ajpgi.00473.2006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K(+) channels, K(v)1.1 and K(v)2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits K(v)2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of K(v)1.2, a major delayed rectifier K(+) channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited K(v)1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of K(v)1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on K(v)2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to K(v)1.2 in an expression system, open-form Syn-1A failed to reduce K(v)1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on K(v)2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different K(v) channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types.
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Affiliation(s)
- Leila Neshatian
- Department of Medicine and Physiology, University Health Network, University of Toronto, Toronto, Canada
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49
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Abstract
The regulation of ion channels involves more than just modulation of their synthesis and kinetics, as controls on their trafficking and localization are also important. Although the body of knowledge is fairly large, the entire trafficking pathway is not known for any one channel. This review summarizes current knowledge on the trafficking of potassium channels that are expressed in the heart. Our knowledge of channel assembly, trafficking through the Golgi apparatus and on to the surface is covered, as are controls on channel surface retention and endocytosis.
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Affiliation(s)
- David F Steele
- Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3
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50
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Singer-Lahat D, Sheinin A, Chikvashvili D, Tsuk S, Greitzer D, Friedrich R, Feinshreiber L, Ashery U, Benveniste M, Levitan ES, Lotan I. K+ channel facilitation of exocytosis by dynamic interaction with syntaxin. J Neurosci 2007; 27:1651-8. [PMID: 17301173 PMCID: PMC6673747 DOI: 10.1523/jneurosci.4006-06.2007] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca2+. This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca2+. However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.
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Affiliation(s)
- Dafna Singer-Lahat
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Anton Sheinin
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Dodo Chikvashvili
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Sharon Tsuk
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Dafna Greitzer
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Reut Friedrich
- Department of Neurobiochemistry, Life Sciences Institute, Tel-Aviv University, 69978 Ramat-Aviv, Israel
| | - Lori Feinshreiber
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
| | - Uri Ashery
- Department of Neurobiochemistry, Life Sciences Institute, Tel-Aviv University, 69978 Ramat-Aviv, Israel
| | - Morris Benveniste
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
- Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia 30310
| | - Edwin S. Levitan
- Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and
| | - Ilana Lotan
- Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
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