title: Ubiquitin ligase Nedd4-2 modulates Kv1.3 current amplitude and ion channel protein targeting.
abstract: Voltage-dependent potassium channels (Kv) go beyond the stabilization of the resting potential and regulate biochemical pathways, regulate intracellular signaling, and detect energy homeostasis. Because targeted deletion and pharmacological block of the Kv1.3 channel protein produce marked changes in metabolism, resistance to diet-induced obesity, and changes in olfactory structure and function, this investigation explored Nedd4-2-mediated ubiquitination and degradation to regulate Kv1.3 channel density. Heterologous coexpression of Nedd4-2 ligase and Kv1.3 in HEK 293 cells reduced Kv1.3 current density without modulation of kinetic properties as measured by patch-clamp electrophysiology. Modulation of current density was dependent on ligase activity and was lost through point mutation of cysteine 938 in the catalytic site of the ligase (Nedd4-2CS). Incorporation of adaptor protein Grb10 relieved Nedd4-2-induced current suppression as did application of the proteasome inhibitor Mg-132. SDS-PAGE and immunoprecipitation strategies demonstrated a channel/adaptor/ligase signalplex. Pixel immunodensity was reduced for Kv1.3 in the presence of Nedd4-2, which was eliminated upon additional incorporation of Grb10. We confirmed Nedd4-2/Grb10 coimmunoprecipitation and observed an increased immunodensity for Nedd4-2 in the presence of Kv1.3 plus Grb10, regardless of whether the catalytic site was active. Kv1.3/Nedd4-2 were reciprocally coimmunoprecipated, whereby mutation of the COOH-terminal, SH3-recognition (493-498), or ubiquitination sites on Kv1.3 (lysines 467, 476, 498) retained coimmunoprecipitation, while the latter prevented the reduction in channel density. A model is presented for which an atypical interaction outside the canonical PY motif may permit channel/ligase interaction to lead to protein degradation and reduced current density, which can involve Nedd4-2/Grb10 interactions to disrupt Kv1.3 loss of current density.
authors.affiliation: Program in Neuroscience, Florida State University, Tallahassee, Florida;
authors.forename: Patricio
authors.initials: P
authors.lastname: Vélez
authors.affiliation: Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida; and.
authors.forename: Austin B
authors.initials: AB
authors.lastname: Schwartz
authors.affiliation: Program in Neuroscience, Florida State University, Tallahassee, Florida;
authors.forename: Subashini R
authors.initials: SR
authors.lastname: Iyer
authors.affiliation: Department of Biological Sciences, Florida State University, Tallahassee, Florida.
authors.forename: Anthony
authors.initials: A
authors.lastname: Warrington
authors.affiliation: Program in Neuroscience, Florida State University, Tallahassee, Florida; Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida; and Department of Biological Sciences, Florida State University, Tallahassee, Florida dfadool@bio.fsu.edu.
authors.forename: Debra Ann
authors.initials: DA
authors.lastname: Fadool
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002461:Cell Line, Transformed; D003545:Cysteine / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000235:genetics*; D004558:Electric Stimulation; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D051383:GRB10 Adaptor Protein / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D051664:Kv1.3 Potassium Channel / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D000075702:Nedd4 Ubiquitin Protein Ligases; D018408:Patch-Clamp Techniques; D034702:RNA-Binding Protein FUS / Q000276:immunology; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics
chemical_list: D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D056827:Endosomal Sorting Complexes Required for Transport; D051664:Kv1.3 Potassium Channel; D007976:Leupeptins; D034702:RNA-Binding Protein FUS; D051383:GRB10 Adaptor Protein; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine; D003545:Cysteine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: adaptor protein; modulation; olfactory; potassium channel; ubiquination