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.
Clayton
Cecilea C
CC
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Xu
Mei
M
Chavkin
Charles
C
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