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Thiol‐Oxidation Impairs Alpha2‐Adrenergic Receptor Mediated Inhibition of N‐type Ca 2+ channels and Results in Receptor Mislocalization
Author(s) -
Mui Ryan K,
Fernandes Roxanne N,
Feng Huijie,
Galligan James J
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.991.14
Subject(s) - chemistry , oxidative stress , receptor , apocynin , nadph oxidase , pertussis toxin , transfection , hek 293 cells , biophysics , g protein , microbiology and biotechnology , biochemistry , biology , gene
Oxidative stress and sympathetic hyperactivity contribute to hypertension. In sympathetic neurons, activation of alpha2‐adrenergic receptors (α2AR) decreases nerve terminal release of norepinephrine (NE) by activating Gαi/o proteins to inhibit N‐type voltage‐gated Ca 2+ channels. We have shown in DOCA‐salt hypertensive rats that α2AR‐mediated inhibition of Ca 2+ channels is impaired in sympathetic neurons, resulting in increased NE release. Furthermore, blood pressure and α2AR function are preserved following treatment with clodronate (macrophage depletion agent) and apocynin (NADPH oxidase inhibitor). Oxidative stress is associated with adrenergic dysfunction and Gαi/o are targets of oxidative stress. Inhibition of α2AR‐Gαi/o coupling following pertussis toxin (PTX) treatment occurs due to ADP‐ribosylation on the C‐terminal cysteine (position 351) of Gαi/o. It is not known if thiol‐oxidation of this residue results in similar uncoupling. Therefore, we tested the hypothesis that oxidizing agents impair α2‐AR function by thiol‐oxidation of Cys351 on Gαi/o. Methods These studies used cultured sympathetic neurons, G1A1 cells (HEK cells stably expressing N‐type Ca 2+ channels), and HEK 293T cells. Cell lines were co‐transfected with α2AR and either WT‐Gαo or PTX‐insensitive Gαo (PTXi‐Gαo, Cys351Gly). Oxidative stress was induced with H 2 O 2 , t‐BOOH, or diamide (thiol‐specific oxidant). Whole cell patch clamp was used to detect inhibition of Ca 2+ current using NE to activate α2AR and GppNHp to inhibit Ca 2+ current independent of α2AR activation. Immunohistochemistry was used to localize α2AR and Gαo following oxidant treatment before and after activation of α2AR with NE. Results In sympathetic neurons, α2AR‐mediated inhibition of Ca 2+ current was reduced following H 2 O 2 , t‐BOOH or diamide treatment vs. untreated neurons. GppNHp inhibited Ca 2+ current equally in all neurons independent of oxidative stress. Oxidative stress impaired α2AR‐mediated inhibition of Ca 2+ current similarly in G1A1 cells co‐transfected with WT‐Gαo or PTXi‐Gαo. Oxidative stress had no effect on cell capacitance or Ca 2+ channel activity in neurons and G1A1 cells. In HEK 293T cells, oxidative stress did not affect co‐localization of α2AR and Gαo, however, oxidative stress resulted in greater cytoplasmic localization of α2AR. Conclusions Thiol‐oxidation results in impaired α2AR‐mediated inhibition of Ca 2+ current. Thiol‐oxidation does not affect Gαo coupling to α2AR. Dysfunction may be attributed to thiol‐oxidation of cysteine residues on α2AR, which do not affect Gαo coupling, but may alter structural motifs that could affect receptor localization to the cell membrane. Support or Funding Information Supported by NIH 2P01HL070687 and American Heart Association (15PRE25090218)