title: Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking.
abstract: The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport.
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Martin N
authors.initials: MN
authors.lastname: Andersen
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Louise L
authors.initials: LL
authors.lastname: Hefting
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Annette B
authors.initials: AB
authors.lastname: Steffensen
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schmitt
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Søren-Peter
authors.initials: SP
authors.lastname: Olesen
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Alicia
authors.initials: A
authors.lastname: Lundby
authors.affiliation: The Danish National Research Foundation Center for Cardiac Arrhythmia and The Faculty of Health and Medical Sciences, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; and hannebr@sund.ku.dk.
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology*; D022161:Transport Vesicles / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D051657:KCNQ1 Potassium Channel; D047428:Protein Kinase Inhibitors; D000075702:Nedd4 Ubiquitin Protein Ligases; D044767:Ubiquitin-Protein Ligases; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: KCNQ1; basolateral targeting; cell signaling; epithelia; ubiquitylation