title: O-glycosylation of the cardiac I(Ks) complex.
abstract: Post-translational modifications of the KCNQ1–KCNE1 (Kv7) K+ channel complex are vital for regulation of the cardiac IKs current and action potential duration. Here, we show the KCNE1 regulatory subunit is O-glycosylated with mucin-type glycans in vivo. As O-linked glycosylation sites are not recognizable by sequence gazing, we designed a novel set of glycosylation mutants and KCNE chimeras and analysed their glycan content using deglycosylation enzymes. Our results show that KCNE1 is exclusively O-glycosylated at Thr-7, which is also required for N-glycosylation at Asn-5. For wild type KCNE1, the overlapping N- and O-glycosylation sites are innocuous for subunit biogenesis; however, mutation of Thr-7 to a non-hydroxylated residue yielded mostly unglycosylated protein and a small fraction of mono-N-glycosylated protein. The compounded hypoglycosylation was equally deleterious for KCNQ1–KCNE1 cell surface expression, demonstrating that KCNE1 O-glycosylation is a post-translational modification that is integral for the proper biogenesis and anterograde trafficking of the cardiac IKs complex. The enzymatic assays and panel of glycosylation mutants used here will be valuable for identifying the different KCNE1 glycoforms in native cells and determining the roles N- and O-glycosylation play in KCNQ1–KCNE1 function and localization in cardiomyocytes,
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Kshama D
authors.initials: KD
authors.lastname: Chandrasekhar
authors.forename: Anatoli
authors.initials: A
authors.lastname: Lvov
authors.forename: Cecile
authors.initials: C
authors.lastname: Terrenoire
authors.forename: Grace Y
authors.initials: GY
authors.lastname: Gao
authors.forename: Robert S
authors.initials: RS
authors.lastname: Kass
authors.forename: William R
authors.initials: WR
authors.lastname: Kobertz
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; D011134:Polysaccharides; D024642:Potassium Channels, Voltage-Gated; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C059983:potassium channel protein I(sk); D013912:Threonine; D001216:Asparagine