title: S-glutathionylation of an auxiliary subunit confers redox sensitivity to Kv4 channel inactivation.
abstract: Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast "ball-and-chain" N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a). Here, we report that both DPP6a and DPP10a, like Kv subunits with redox-sensitive N-type inactivation, contain a highly conserved cysteine in their N-termini (Cys-13). To test if N-type inactivation mediated by DPP6a or DPP10a is redox sensitive, Xenopus oocyte recordings were performed to examine the effects of two common oxidants, tert-butyl hydroperoxide (tBHP) and diamide. Both oxidants markedly modulate DPP6a- or DPP10a-conferred N-type inactivation of Kv4 channels, slowing the overall inactivation and increasing the peak current. These functional effects are fully reversed by the reducing agent dithiothreitol (DTT) and appear to be due to a selective modulation of the N-type inactivation mediated by these auxiliary subunits. Mutation of DPP6a Cys-13 to serine eliminated the tBHP or diamide effects, confirming the importance of Cys-13 to the oxidative regulation. Biochemical studies designed to elucidate the underlying molecular mechanism show no evidence of protein-protein disulfide linkage formation following cysteine oxidation. Instead, using a biotinylated glutathione (BioGEE) reagent, we discovered that oxidation by tBHP or diamide leads to S-glutathionylation of Cys-13, suggesting that S-glutathionylation underlies the regulation of fast N-type inactivation by redox. In conclusion, our studies suggest that Kv4-based A-type current in neurons may show differential redox sensitivity depending on whether DPP6a or DPP10a is highly expressed, and that the S-glutathionylation mechanism may play a previously unappreciated role in mediating excitability changes and neuropathologies associated with ROS.
authors.affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Henry H
authors.initials: HH
authors.lastname: Jerng
authors.affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Pfaffinger
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003958:Diamide / Q000494:pharmacology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051672:Shal Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014982:Xenopus laevis; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: C496749:KCNIP3 protein, human; D051605:Kv Channel-Interacting Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D051672:Shal Potassium Channels; D003958:Diamide; D020122:tert-Butylhydroperoxide; C503535:DPP6 protein, human; C476879:DPP10 protein, human; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D005978:Glutathione; D004229:Dithiothreitol