anisomycin and Neurodegenerative-Diseases

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(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 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(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.

Topics: Animals; Anisomycin; Antibodies; Behavior; Blotting, Western; Cell Line; Cell Line, Tumor; CHO Cells; Cricetinae; DNA; Dose-Response Relationship, Drug; Electrophysiology; Enzyme-Linked Immunosorbent Assay; Female; G Protein-Coupled Inwardly-Rectifying Potassium Channels; GTP-Binding Proteins; Heart Atria; Heart Ventricles; Humans; Immunoblotting; Immunohistochemistry; Inflammation; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Microscopy, Fluorescence; Muscle Cells; Neurodegenerative Diseases; Neurons; NIH 3T3 Cells; Phosphorylation; Plasmids; Protein Structure, Tertiary; Sciatic Nerve; Spinal Cord; Stress, Physiological; Tyrosine; Xenopus laevis