Tyrosine Phosphorylation of Kir3.1 in Spinal Cord Is Induced by Acute Inflammation, Chronic Neuropathic Pain, and Behavioral Stress

Danielle L Ippolito,Mei Xu,Michael R Bruchas,Kevin Wickman,Charles Chavkin

doi:10.1074/jbc.m507069200

Danielle L Ippolito, Mei Xu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m507069200

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Abstract

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.

Highlights

Kir3 channels play an important role in regulating cardiac and neuronal signaling [1,2,3,4], modulation of channel function mediated by tyrosine phosphorylation could influence cardiac and CNS excitability
To identify physiological stimuli promoting Kir3 tyrosine phosphorylation in the spinal cord, in this study we developed an antibody selective for Kir3.1 when phosphorylated at tyrosine 12, a residue located in the cytoplasmic N-terminal domain
After characterizing pY12-Kir3.1 specificity and phosphoselectivity in primary cardiac myocyte cultures and transfected cell lines, we evaluated phosphorylation of Tyr12-Kir3.1 in spinal cord slices from mice subjected to hindpaw formalin injection or sciatic nerve ligation, models of inflammatory and neuropathic pain, respectively

Summary

Introduction

Kir channels play an important role in regulating cardiac and neuronal signaling [1,2,3,4], modulation of channel function mediated by tyrosine phosphorylation could influence cardiac and CNS excitability. Recent studies in mice with genetically ablated Kir3.1 have shown that Kir plays a role in attenuating opioid-mediated antinociception by activating heterotetramers of Kir3.1 and Kir3.2 in the dorsal horn of the spinal cord [4, 5]. Because tyrosine kinases are up-regulated and activated in animal models of spinally mediated acute and chronic pain [11], it is reasonable to hypothesize that Kir may be phosphorylated at N-terminal tyrosine residues in response to these stimuli. We further investigated pY12-Kir3.1 in a mouse model of chronic stress to determine whether Kir3.1 Tyr phosphorylation occurred in the dorsal horn in response to stressful stimuli independently of nociception. This study provides evidence that Kir3.1 tyrosine phosphorylation occurs in response to nociceptive stimuli and physiological stress

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