Abstract
Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K(ir)3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K(ir)3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wild-type mice, but not in mice lacking the kappa-opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K(ir)3.1-ir. The U50,488-induced increase in pY12-K(ir)3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K(ir)3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K(ir)3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K(ir)3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K(ir)3.1-ir; and the heterologous desensitization of K(ir)3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K(ir)3.1 and channel inhibition through a GRK3-, p38 MAPK- and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.
Highlights
EXPERIMENTAL PROCEDURESChemicals—Norbinaltorphimine HCl and (-) U50,488 were obtained from Tocris (Ellisville, MO) and the NIDA Drug Supply Program
USPHS Grants R37-DA11672 and K05 DA20570 from NIDA. 1 To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357280, 1959 Pacific Ave
Release of the G␥ subunit following activation of the Gi/o class of G-protein-coupled receptors (GPCRs) directly activates Kir3 channels, and channel deactivation occurs when the GTP bound to G␣ is hydrolyzed to GDP, and G␥ dissociates from the channel [2, 3]
Summary
Chemicals—Norbinaltorphimine HCl and (-) U50,488 were obtained from Tocris (Ellisville, MO) and the NIDA Drug Supply Program. KOR(S369A)-GFP, does not recruit -arrestin [16] and does not internalize or desensitize after agonist exposure [10] Stimulation of this mutant receptor with U50,488 failed to increase pY12-Kir3.1-ir at any time point tested; the lack of effect at 60 min is shown (Fig. 3D). 10 M U50,488 pretreatment of KOR(S369)-GFP expressing AtT-20 cells did not significantly affect subsequent somatostatin evoked potassium currents (Fig. 6C) These results suggest that KOR-mediated phosphorylation of Kir3.1 results in reduced channel conductance and that this effect can be blocked by inhibiting p38 MAP kinase. Quantitation of pixel intensity changes in replicate experor JNK (21, 26 –28) In both types of cells somatostatin was able iments confirmed that phospho-Src-ir was significantly to induce inward potassium currents that were unaffected by increased by 10 M U50,488 treatment of KOR-GFP expressing.
Sign-in/Register to view highlights & summary 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.
