Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCε-mediated phosphorylation at S800
Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCε-mediated phosphorylation at S800
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DOI：
10.1016/j.pain.2006.02.016
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发表时间：
2006-07-01
期刊：
影响因子：
7.4
通讯作者：
Tominaga, Makoto
中科院分区：
文献类型：
--
作者：
Mandadi, Sravan;Tominaga, 'Tomoko;Tominaga, Makoto
调节TRPV 1介导的伤害感受的抑制和易化作用的重要机制分别是脱敏和磷酸化。使用钙离子成像，我们以前已经表明，脱敏TRPV 1后，连续辣椒素应用逆转蛋白激酶C激活背根神经节神经元和CHO细胞。在这里，使用钙离子成像和膜片钳方法，我们表明，PMA诱导的PKC β激活是必不可少的敏感性增加的脱敏TRPV 1。TRPV 1有两个推定的底物S502和S800用于PKC ε介导的磷酸化。膜片钳分析表明，单突变体S502 A或S800 A对脱敏TRPV 1敏感性增加的贡献与双突变体S502 A/S800 A中观察到的无区别。由于S502是通过激酶如PKC、PKA或CAMK II磷酸化TRPV 1的非特异性底物，因此研究了PKC特异性底物S800的作用的证据。首次证明了TRPV 1在S800的体内磷酸化的证据。我们还表明，PKC的表达水平增加了磷酸化TRPV 1蛋白的量，使用特异性磷酸化TRPV 1在S800。此外，抗磷酸化TRPV 1抗体检测到TRPV 1在小鼠和大鼠DRG神经元中的磷酸化，并且可能用于关于天然组织中的伤害感受的研究。因此，这项研究确定了PKC β和S800作为重要的治疗靶点，可能有助于调节对TRPV 1的抑制作用，从而使其脱敏。(c)2006年国际疼痛研究协会。Elsevier B. V.出版，保留所有权利。
Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKC epsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKC epsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKC epsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKC epsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization. (c) 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.