Prior studies showed that tyrosine-12 phosphorylation in the amino terminal, cytoplasmic domain of the G-Protein gated inwardly rectifying potassium channel, Kir3.1 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-Kir3.1-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-Kir3.1-ir. The U50,488-induced increase in pY12-Kir3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-Kir3.1-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 Kir3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of Kir3.1 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-Kir3.1-ir; and the heterologous desensitization of Kir3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-Kir3.1 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.
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