Novakova-Tousova K, Vyklicky L, Susankova K, Benedikt J, Samad A, Teisinger J, Vlachova V
Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
Neuroscience. 2007 Oct 12;149(1):144-54. doi: 10.1016/j.neuroscience.2007.07.039. Epub 2007 Aug 8.
Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.
激动剂诱导的瞬时受体电位香草酸受体1（TRPV1）脱敏是缓解神经性和炎性疼痛的关键策略之一。该过程由TRPV1受体激活引发，随后细胞外Ca(2+)通过该通道进入感觉神经元。负责TRPV1脱敏的一个突出机制是Ca(2+)/钙调蛋白依赖性酶磷酸酶2B（钙调神经磷酸酶）使TRPV1蛋白去磷酸化。在目前已确定的几个共有磷酸化位点中，最显著的是两个Ca(2+)/钙调蛋白依赖性激酶II（CaMKII）位点，磷酸化和去磷酸化状态之间的动态平衡可能调节激动剂结合。我们使用全细胞膜片钳技术，在表达大鼠TRPV1野生型或CaMKII磷酸化位点突变体的人胚肾（HEK）293T细胞中研究了急性Ca(2+)依赖性脱敏的机制。非磷酸化突变体S502A/T704I对辣椒素不敏感，但S502A/T704A构建体功能完全正常，表明704位需要特定残基。附近保守残基R701的点突变强烈影响热、辣椒素和pH诱发的电流。由于该残基构成一个严格的CaMKII共有位点，但预计也参与与膜磷脂酰肌醇4,5-二磷酸（PIP(2)）的相互作用，这些数据表明，除了去磷酸化或作为其结果外，与由R701和T704组成的瞬时受体电位框相邻的短C末端近膜段可能参与脱敏TRPV1通道的门控动力学减慢。
