Tyrosine phosphorylation of K_ir3.1 in spinal cord is induced by acute inflammation, chronic neuropathic pain, and behavioral stress

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K_ir3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K_ir3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K_ir3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K_ir3.1 phosphorylation in cultured mammalian cell and spinal cord. The antibody was found to discriminate between the phospho-Tyr ¹² of K_ir3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K_ir3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K _ir3.1 channels. Mice lacking K_ir3.1 following targeted gene disruption did not show specific pY12-K_ir3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K_ir3.1 in the dorsal horn. This study provides evidence that K_ir3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K_ir3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.