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.
Highlights
Many voltage-dependent potassium (Kv) channels inactivate in response to prolonged depolarization
To determine if N-type inactivation mediated by DPP6a and DPP10a is likely redox sensitive, we aligned their N-terminal sequences to look for conserved cysteine residues and discovered a highly conserved cysteine at position 13 (Cys-13) for both DPP6a and DPP10a (Fig. 1B)
Regulation of DPP6a-mediated N-type inactivation tertbutyl hydroperoxide, an analog of the native oxidant hydrogen peroxide To test whether DPP6a- and DPP10a-mediated N-type inactivation is sensitive to redox, DPP6a was co-expressed with Kv4.2 and KChIP3a in Xenopus oocytes, and whole-oocyte currents were elicited by families of step depolarizations under the voltage-clamp configuration
Summary
Many voltage-dependent potassium (Kv) channels inactivate in response to prolonged depolarization. Fast inactivation is often produced by a ‘‘balland-chain’’ mechanism, where a cytoplasmic N-terminal segment enters and occludes the inner pore during channel opening, thereby terminating K+ conduction [2,3]. This ‘‘N-type’’ inactivation can be mediated by N-terminal sequences contained on the pore-forming subunits or on certain Kv channel auxiliary subunits [2,4,5,6,7,8,9,10,11,12,13]. The importance of disulfide bridge formation for this effect remains unclear, since oxidants can generate reaction intermediates and by-products in addition to inducing disulfides that might affect N-type inactivation. The balance between the formation of mixed protein-glutathione disulfides verses protein-protein disulfides depends on two factors: the relative redox potentials between cysteine thiols and GSH and the relative concentrations of reactant and product species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
