Stein Alexander T, Ufret-Vincenty Carmen A, Hua Li, Santana Luis F, Gordon Sharona E
Graduate Program in Neurobiology and Behavior, University of Washington, Seattle, WA 98195, USA.
J Gen Physiol. 2006 Nov;128(5):509-22. doi: 10.1085/jgp.200609576.
Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.
痛觉转导离子通道TRPV1的敏化是神经生长因子（NGF）等促痛剂引起热痛觉过敏的基础。目前被广泛接受的模型是，痛觉过敏期间NGF介导的TRPV1功能增强是利用磷脂酶C（PLC）的激活来裂解PIP2，而PIP2被认为可持续性抑制TRPV1。在本研究中，我们对PLC模型进行了测试，发现了两条直接挑战其有效性的证据：（1）聚赖氨酸，一种阳离子磷酸肌醇螯合剂，抑制而非增强TRPV1；（2）将PIP2直接应用于内向外膜片可显著增强TRPV1。此外，我们展示了四类实验，表明PI3K在物理和功能上与TRPV1偶联：（1）在酵母双杂交实验中，PI3K的p85β亚基与TRPV1的N端区域相互作用；（2）PI3K-p85β与TRPV1在HEK293细胞和背根神经节（DRG）神经元中共同免疫沉淀；（3）TRPV1在体外与重组PI3K-p85相互作用；（4）PI3K的特异性抑制剂渥曼青霉素完全消除了急性分离的DRG神经元中NGF介导的敏化。最后，同时进行的电生理和全内反射荧光（TIRF）显微镜记录表明，NGF增加了质膜上通道的数量。我们提出了一种新的NGF介导的痛觉过敏模型，其中TRPV1和PI3K在信号转导复合物中的物理偶联促进了TRPV1向质膜的转运。
