Tyrosine phosphorylation of Kir3 following kappa-opioid receptor activation of p38 MAPK causes heterologous desensitization.
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.
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Cecilea C
CC
Clayton
Mei
M
Xu
Charles
C
Chavkin
D000818:Animals; D004594:Electrophysiology; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000502:physiology*; D015151:Immunoblotting; D007473:Ion Channels; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000502:physiology*; D012584:Sciatic Nerve / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007473:Ion Channels; D017473:Receptors, Opioid, kappa; D014443:Tyrosine; D054770:G-Protein-Coupled Receptor Kinase 3; C517850:GRK3 protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases