Oxidation modulates LINGO2-induced inactivation of large conductance, Ca 2+ -activated potassium channels

• Published in: The Journal of biological chemistry 2023-03, Vol.299 (3), p.102975
• Main Authors: Dudem, Srikanth, Boon, Pei Xin, Mullins, Nicholas, McClafferty, Heather, Shipston, Michael J, Wilkinson, Richard D A, Lobb, Ian, Sergeant, Gerard P, Thornbury, Keith D, Tikhonova, Irina G, Hollywood, Mark A
• Format: Article
• Language: English
• Subjects: Calcium - metabolism; Cysteine - metabolism; Large-Conductance Calcium-Activated Potassium Channels - genetics; Large-Conductance Calcium-Activated Potassium Channels - metabolism; Methionine - metabolism; Oxidation-Reduction; Peptides - metabolism
• ISSN: 1083-351X

Ca and voltage-activated K (BK) channels are ubiquitous ion channels that can be modulated by accessory proteins, including β, γ, and LINGO1 BK subunits. In this study, we utilized a combination of site-directed mutagenesis, patch clamp electrophysiology, and molecular modeling to investigate if the biophysical properties of BK currents were affected by coexpression of LINGO2 and to examine how they are regulated by oxidation. We demonstrate that LINGO2 is a regulator of BK channels, since its coexpression with BK channels yields rapid inactivating currents, the activation of which is shifted ∼-30 mV compared to that of BKα currents. Furthermore, we show the oxidation of BK:LINGO2 currents (by exposure to epifluorescence illumination or chloramine-T) abolished inactivation. The effect of illumination depended on the presence of GFP, suggesting that it released free radicals which oxidized cysteine or methionine residues. In addition, the oxidation effects were resistant to treatment with the cysteine-specific reducing agent DTT, suggesting that methionine rather than cysteine residues may be involved. Our data with synthetic LINGO2 tail peptides further demonstrate that the rate of inactivation was slowed when residues M603 or M605 were oxidized, and practically abolished when both were oxidized. Taken together, these data demonstrate that both methionine residues in the LINGO2 tail mediate the effect of oxidation on BK:LINGO2 channels. Our molecular modeling suggests that methionine oxidation reduces the lipophilicity of the tail, thus preventing it from occluding the pore of the BK channel.