2001 Fiscal Year Final Research Report Summary
Mechanism of nociception : Structure-function relationship of capsaicin receptor and analysis of its regulation mechanism
| Project/Area Number |
12670037
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Mie University (2001)
University of Tsukuba (2000) |
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Principal Investigator |
TOMINAGA Makoto Mie University, Faculty of Medicine, Professor, 医学部, 教授 (90260041)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Pain / capsaicin receptor / VR1 / phosphorylation / ATP / P2Y_1 receptor / PKC / PKC |
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| Research Abstract |
Tissue damage associated with infection, inflammation, or ischemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit pain at the site of injury. An important component of this pro-algesic response is ATP released from different cell types. Extracellular ATP excites the nociceptive endings of nearby sensory nerves, evoking a sensation of pain. To address whether metabotropic P2Y receptors are involved in VR1-mediated nociceptive responses, the effects of extracellular ATP on VR1 expressed in HEK293 cells and rat DRG neurons were examined. In cells expressing VR1, extracellular ATP increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y_1 receptors in a PKC-dependent pathway. In the presence of ATP, the temperature threshold for VR1 activation was reduced from 42 ℃ to 35 ℃, such that normally non-painful thermal stimuli (i.e. normal body temperature) were capable of activating VR1. This represents a novel mechanism through which ATP might cause pain in a pathway distinct from the activation of P2X receptors.
Next, it has to be addressed whether VR1 is directly phosphorylated by PKC and if so which amino acid residues are involved in the phosphorylation. Direct phosphorylation of VR1 upon application of PMA was proven biochemically in cells expressing VR1. An in vitro kinase assay using GST fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxy terminal of VR1 were phosphorylated by PKCε. Patch-clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites in VR1 showed that two Ser residues were involved in the potentiation of the currents evoked by either PMA or ATP. These two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.
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