Wiedmann F , Schlund D , Faustino F , Kraft M , Ratte A , Thomas D , Katus HA , Schmidt C .

Excellence grant German Center for Cardiovascular Research, Rahel Goitein-Straus Scholarship and Olympia-Morata Scholarship University of Heidelberg, Faculty of Medicine, F/41/15 and F/35/18 German Heart Foundation /German Foundation of Heart Research, SCHM 3358/1-1 to C.S Deutsche Forschungsgemeinschaft, Kaltenbach Scholarship German Heart Foundation/German Foundation of Heart Research, Otto-Hess-Scholarship and the Research-Scholarship German Cardiac Society

	Figure 1. hTREK-1 channels harbor two putative N-glycosylation sites. (a) Two potential N-glycosylation consensus sites (yellow), consisting of an asparagine residue (N, red) followed by any amino acid except proline (x) and a serine or threonine residue (S/T, red) can be found in the extracellular part of the hTREK-1 amino acid sequence. Asparagine residues possibly modified by carbohydrates are located in positions 110 and 134. (b) Depicts a schematic membrane topology model of a hTREK-1 channel monomer consisting of two pore-forming loops (P1 and P2) surrounded by four transmembrane domains (M1–M4; top: extracellular space, bottom: intracellular space). Both potential N-glycosylation motifs are located in the extracellular M1-P1 interdomain. (c) Three-dimensional model of a hTREK-1 channel dimer, based on its crystal structure (PDB ID: 4TWK) [40,41,42]. N110 is located at the top of the overhead domain, and N134 is situated at a more lateral position. (d) A partial sequence alignment comparing hTREK-1 protein sequences of different species showing conservation of both motifs (‘*’, full conservation; ‘:’, conservative substitution; ‘.’, semiconservative substitution). (e) Immunoblot of hTREK-1-1d4 channel subunits heterologously expressed in Xenopus laevis oocytes. After coinjection of the antibiotic N-glycosylation inhibitor tunicamycin (TM), hTREK-1-1d4 proteins display increased electrophoretic mobility. The signals of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are provided as loading controls.
	Figure 2. Inhibition of N-glycosylation by tunicamycin decreases TREK-1 currents. (a) The TREK-1 currents elicited by a 500 ms depolarizing voltage step from −80 mV to +20 mV (see depicted pulse protocol) are displayed under control conditions (CTRL) and 48 h after administration of TM by incubation (left) or cytoplasmic injection (right). Representative recording of n = 6–12 cells. (b) The mean potassium currents at +20 mV (top) and the resting membrane potentials (RMPs, bottom) are provided for different time points (24 h, black bars; 48 h, white bars) after TM incubation (left; n = 3–15) or cytoplasmic injection (right; n = 3–15 cells). (c) Representative families of macroscopic hTREK-1 potassium currents recorded from Xenopus laevis oocytes by application of the depicted pulse protocol. (d) Corresponding mean step current amplitudes plotted as functions of the test pulse potential showing comparable current-voltage relationships under control conditions and after application of TM. Inserts: the data are presented relative to the maximum current amplitude measured at +60 mV. The data are given as the mean ± standard error of the mean (SEM). The zero-current levels are indicated by dashed lines. The pulse protocols are depicted below the respective current traces. * p < 0.05, ** p < 0.01.
	Figure 3. Molecular biological disruption of hTREK-1 N-glycosylation. (a) N-glycosylation can be disrupted either by introducing a proline residue in position +3 relative to the N-glycosylation acceptor residue (a,c,e,g,i) or by substituting the carbohydrate acceptor asparagine to glutamine (b,d,f,h,j). (a,b) Protein lysates of Xenopus laevis oocytes injected with RNA of the indicated hTREK-1-1d4 mutant constructs lacking the first, the second or both the first and second N-glycosylation motifs were subjected to immunoblotting. Changes in carbohydrate modifications are displayed as altered electrophoretic mobility. Please note that the E113P substitution resulted in incomplete disruption of N-glycosylation at N110. The immunosignals of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are given as loading controls. (c,d) Representative current traces recorded from Xenopus laevis oocytes expressing the indicated hTREK-1 variants after 24 h (left) or 48 h (right). The currents were evoked by the application of depolarizing voltage steps from −80 mV to +20 mV. Representative current traces of n = 4–12 cells are shown. The mean current amplitudes (top) and resting membrane potentials (bottom) of the cells included in this experiment are displayed in (e,f). (g,h) Families of hTREK-1 current traces evoked by the displayed pulse step protocols 48 h after injection of the hTREK-1 WT or mutant construct. (i,j) Activation curves recorded under isochronal conditions 48 h after RNA injection. Inserts: the data presented relative to the maximum current amplitude measured at +60 mV display comparable voltage-current relationships between glycosylated and nonglycosylated hTREK-1 channel subunits. The data are provided as the mean ± standard error of the mean (SEM). The dashed lines indicate the zero-current levels. The pulse protocols are depicted next to the current traces (* p < 0.05, ** p < 0.01, *** p < 0.001).
	Figure 4. N-glycosylation of hTREK-1 currents, expressed in mammalian cells. To determine whether hTREK-1 channel subunits undergo N-glycosylation in mammalian cells, the relevance of the hTREK-1 N-glycosylation sites N110 and N134 was confirmed in HEK-293T cells. (a–c) Protein lysates of HEK-293T cells expressing hTREK-1-1d4 WT or mutant constructs were subjected to anti 1d4-immunoblotting under control conditions, after administration of tunicamycin (TM) or after cleavage of N-linked carbohydrates by the N-glycosidase PNGase F, as indicated by (+) or (−). Changes in carbohydrate modifications are displayed as altered electrophoretic mobility, and immunoblots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are provided as loading controls.
	Figure 5. N-glycosylation regulates the surface expression of hTREK-1 channels. (a) EGFP-reporter coupled WT hTREK-1 channel subunits or glutamine mutants lacking either one or both N-glycosylation motifs were expressed in HeLa cells. Cell membranes stained with Alexa Fluor 594-labeled wheat germ agglutinin are depicted in red. The fluorescence signals of hTREK-1-eGFP variants are shown in green. The overlays (yellow) demonstrate colocalization of diglycosylated, monoglycosylated and nonglycosylated double-mutant channels with the cellular membrane, and show the preserved surface trafficking of deglycosylated channels. Scale bar: 10 µm. (b) Surface fractions of HEK-293T cells expressing the indicted hTREK-1 N-glycosylation-deficient variants were isolated via surface protein biotinylation, followed by streptavidin precipitation. Immunoblots of the input fractions are displayed on the left, and the mean immunosignals of the surface fractions are given on the right side (n = 3). (c) Ion channel subunit surface fractions (i.e., mean optical densities of the surface blots divided by the input fraction standardized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control relative to the WT signal). The data are provided as the mean ± SEM (* p < 0.05).
	Figure 6. There is no evidence for O-glycosylation of hTREK-1 channels. Immunoblots of hTREK-1 are shown for control conditions, after administration of the O-glycosylation inhibitor benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (BenGal) and after incubation of the protein lysates with a mixture of O-glycosidase and neuraminidase (to yield potential O-glycosides accessible to O-glycosidase), as indicated by (+) and (−). However, no mobility shifts could be observed after treatment with BenGal or O-glycosidase, suggesting a lack of significant O-glycosylation of hTREK-1 in HEK-239T cells. The GAPDH signals are provided as loading controls.
	Figure 4. N-glycosylation of hTREK-1 currents, expressed in mammalian cells. To determine whether hTREK-1 channel subunits undergo N-glycosylation in mammalian cells, the relevance of the hTREK-1 N-glycosylation sites N110 and N134 was confirmed in HEK-293T cells. (a–c) Protein lysates of HEK-293T cells expressing hTREK-1-1d4 WT or mutant constructs were subjected to anti 1d4-immunoblotting under control conditions, after administration of tunicamycin (TM) or after cleavage of N-linked carbohydrates by the N-glycosidase PNGase F, as indicated by (+) or (−). Changes in carbohydrate modifications are displayed as altered electrophoretic mobility, and immunoblots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are provided as loading controls.

Abraham, The two-pore domain potassium channel TREK-1 mediates cardiac fibrosis and diastolic dysfunction. 2018, Pubmed

Abraham, The two-pore domain potassium channel TREK-1 mediates cardiac fibrosis and diastolic dysfunction. 2018, Pubmed
Baycin-Hizal, Physiologic and pathophysiologic consequences of altered sialylation and glycosylation on ion channel function. 2014, Pubmed
Bennett, Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism. 1997, Pubmed
Bennett, Isoform-specific effects of sialic acid on voltage-dependent Na+ channel gating: functional sialic acids are localized to the S5-S6 loop of domain I. 2002, Pubmed
Chandrasekhar, O-glycosylation of the cardiac I(Ks) complex. 2011, Pubmed
Decher, Sodium permeable and "hypersensitive" TREK-1 channels cause ventricular tachycardia. 2017, Pubmed
Decher, Stretch-activated potassium currents in the heart: Focus on TREK-1 and arrhythmias. 2017, Pubmed
Devilliers, Activation of TREK-1 by morphine results in analgesia without adverse side effects. 2013, Pubmed
Dong, K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. 2015, Pubmed
Egenberger, N-linked glycosylation determines cell surface expression of two-pore-domain K+ channel TRESK. 2010, Pubmed , Xenbase
Enyedi, Molecular background of leak K+ currents: two-pore domain potassium channels. 2010, Pubmed
Feliciangeli, The family of K2P channels: salient structural and functional properties. 2015, Pubmed
Fink, Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel. 1996, Pubmed , Xenbase
Freeze, Genetic defects in the human glycome. 2006, Pubmed
Gehrmann, Cardiomyopathy in congenital disorders of glycosylation. 2003, Pubmed
Goldstein, Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels. 2016, Pubmed , Xenbase
Goldstein, Potassium leak channels and the KCNK family of two-P-domain subunits. 2001, Pubmed
Goldstein, International Union of Pharmacology. LV. Nomenclature and molecular relationships of two-P potassium channels. 2005, Pubmed
Goonetilleke, TREK-1 K(+) channels in the cardiovascular system: their significance and potential as a therapeutic target. 2012, Pubmed
Heurteaux, Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype. 2006, Pubmed
Heurteaux, TREK-1, a K+ channel involved in neuroprotection and general anesthesia. 2004, Pubmed
Honoré, The neuronal background K2P channels: focus on TREK1. 2007, Pubmed
Huet, Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells. 2003, Pubmed
Johnson, Isoform-specific effects of the beta2 subunit on voltage-gated sodium channel gating. 2006, Pubmed
Joseph, Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator. 2018, Pubmed
Khanna, Glycosylation increases potassium channel stability and surface expression in mammalian cells. 2001, Pubmed , Xenbase
Kim, Fatty acid-sensitive two-pore domain K+ channels. 2003, Pubmed
Kisselbach, Enhancement of K2P2.1 (TREK1) background currents expressed in Xenopus oocytes by voltage-gated K+ channel β subunits. 2012, Pubmed , Xenbase
Kisselbach, Modulation of K2P 2.1 and K2P 10.1 K(+) channel sensitivity to carvedilol by alternative mRNA translation initiation. 2014, Pubmed , Xenbase
Lamothe, Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility. 2019, Pubmed
Lamothe, Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility. 2018, Pubmed
Lazniewska, Glycosylation of voltage-gated calcium channels in health and disease. 2017, Pubmed
Lazniewska, The "sweet" side of ion channels. 2014, Pubmed
Lesage, TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. 1996, Pubmed , Xenbase
Lesage, Pharmacology of neuronal background potassium channels. 2003, Pubmed
Li Fraine, Dynamic regulation of TREK1 gating by Polycystin 2 via a Filamin A-mediated cytoskeletal Mechanism. 2017, Pubmed
Lolicato, K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. 2017, Pubmed , Xenbase
Maingret, Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. 2000, Pubmed
Maingret, TREK-1 is a heat-activated background K(+) channel. 2000, Pubmed , Xenbase
Mant, N-glycosylation-dependent control of functional expression of background potassium channels K2P3.1 and K2P9.1. 2013, Pubmed
Mellquist, The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency. 1998, Pubmed
Nasr, Identification and characterization of two zebrafish Twik related potassium channels, Kcnk2a and Kcnk2b. 2018, Pubmed
Ohtsubo, Glycosylation in cellular mechanisms of health and disease. 2006, Pubmed
Olvera, Benzyl-2-Acetamido-2-Deoxy-α-d-Galactopyranoside Increases Human Immunodeficiency Virus Replication and Viral Outgrowth Efficacy In Vitro. 2017, Pubmed
Patsos, O-glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines. 2009, Pubmed
Pavel, Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic. 2019, Pubmed
Petrecca, N-linked glycosylation sites determine HERG channel surface membrane expression. 1999, Pubmed
Poupon, Targeting the TREK-1 potassium channel via riluzole to eliminate the neuropathic and depressive-like effects of oxaliplatin. 2018, Pubmed
Schmidt, Upregulation of K(2P)3.1 K+ Current Causes Action Potential Shortening in Patients With Chronic Atrial Fibrillation. 2015, Pubmed
Schmidt, Inverse remodelling of K2P3.1 K+ channel expression and action potential duration in left ventricular dysfunction and atrial fibrillation: implications for patient-specific antiarrhythmic drug therapy. 2017, Pubmed
Schmidt, Stretch-activated two-pore-domain (K2P) potassium channels in the heart: Focus on atrial fibrillation and heart failure. 2017, Pubmed
Sievers, Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. 2011, Pubmed
Tamargo, Pharmacology of cardiac potassium channels. 2004, Pubmed
Thomas, Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium. 2008, Pubmed , Xenbase
Wiedmann, Therapeutic targeting of two-pore-domain potassium (K(2P)) channels in the cardiovascular system. 2016, Pubmed
Wiedmann, Identification of the A293 (AVE1231) Binding Site in the Cardiac Two-Pore-Domain Potassium Channel TASK-1: a Common Low Affinity Antiarrhythmic Drug Binding Site. 2019, Pubmed , Xenbase
Wiedmann, Atrial fibrillation and heart failure-associated remodeling of two-pore-domain potassium (K2P) channels in murine disease models: focus on TASK-1. 2018, Pubmed , Xenbase
Woo, Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition. 2018, Pubmed