This application seeks support for the continuation of a successful grant designed to study the effects of phosphorylation on opioid receptor activation of G-protein coupled potassium channels (Kit3) In the previously funded period, the effects of both serine/threonine phosphorylation and tyrosine phosphorylation on opioid receptor desensitization and Kir3 channel activation were described. We propose to extend these studies by focusing our effort on the specific effects of tyrosine phosphorylation (Y-PO4) on mu opioid receptor (MOR) activation of Kir3. Our previous site directed mutagenesis studies identified potential Y-PO4 I sites within the receptor and channel likely to control the response to opioid agonists. Initially, the mechanisms of this regulation will be studied using cDNA expression of MOR and Kir3.1 in AtT20 cells and primary hippocampal cultures. Studies would be extended by in vitro electrophysiological recording of DAMGO activated Kir3 responses and confocal imaging of receptor and channel trafficking. We would use pharmacological inhibitors to identify the kinases and phosphatases responsible the Y-PO4 mediated effects in these malleable in vitro systems. Results of this aim would test the hypothesis that Y-PO4 of specific sites within MOR and Kir3.1 regulates the efficiency of opioid signaling. The phosphorylation state of specific sites within the MOR and Kir3.1 sequences would be assessed by 32p-incorporation and by probing with novel phosphospecific antibodies. Regulation of opioid receptor signaling by tyrosine phosphorylation has important implications for understanding opioid responses during physiological stress; thus, moving from simple in vitro analyses to more complex, in vivo systems would be a priority. After characterizing the specificity and utility of the phosphospecific antibodies (MOR-YP and Kir3.1-YP) in the in vitro systems, we will test the hypothesis that nerve trauma results in growth factor-induced changes in tyrosine phosphorylation of MOR and Kir3.1 detectable by changes in MOR-YP and Kir3.1-YP immunostaining within nociceptive circuits in spinal cord and brain. Results of the proposed studies are likely to provide additional understanding of the mechanisms mediating the plasticity of opioid signaling.
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