Regulation of sensory TRP channels by phospholipids and G-proteins

最初的资助提案的主题是磷酸肌醇调节热和辣椒素。 敏感的TRPV 1，以及冷敏感和薄荷醇敏感的TRPM 8通道。目前的更新建议 继续研究TRPV 1的磷酸肌醇调节，并解决了一个重要的未解决的问题， 最近在脂质纳米盘中发现了更高分辨率的结构， 辣椒素/香草素结合位点被磷酸肌醇占据，并提出它稳定通道， 静息状态，香草素通过替代脂质激活TRPV 1。然而，PI（4，5）P2和PI（4）P是良好的。 建立了TRPV 1的正辅因子/调节因子，这与该模型难以调和。的确切 然而，磷酸肌醇脂质的性质在结构中没有很好地分辨。在AIM 1中，我们将阐明 香草素结合位点中磷酸肌醇的性质，使用计算建模的组合， 定点诱变、全细胞和切除的贴片电生理学和平面脂质双层。的 TRPM 3离子通道在背根神经节（DRG）神经元中表达;其在小鼠中的遗传缺失导致 对有害热的敏感性改变TRPM 3被热激活，化学激动剂，如 孕烯醇酮硫酸盐（Pregs）和CIM 0216。我们发现，这个通道需要磷酸肌醇， 活性，并且我们还发现磷脂酶C（PLC）偶联受体的激动剂抑制TRPM 3。这 然而，细胞内过量PI（4，5）P2的传递并不能减轻抑制作用，而通过抑制作用的降低， 结合异源三聚体G蛋白βγ亚基（Gβγ汇）的蛋白质。这一发现表明， 在调节TRPM 3中，Gβγ信号转导超过PLC激活。激活不参与的Gi偶联受体 激活PLC也能显著抑制TRPM 3活性，Gβγ汇可减弱这种抑制作用。共表达 在完整细胞中的Gβγ，以及将纯化的Gβγ应用于切除的由内而外的斑块也抑制TRPM 3， 我们通过免疫共沉淀法检测了TRPM 3和Gβ之间的生化相互作用。这些数据表明 Gβγ亚基是TRPM 3的直接负调节因子。我们还发现，内源性Gi- GABAB和阿片受体的偶联抑制了DRG神经元中PregS诱导的Ca ~（2+）信号。在目标2和3中， 我们将测试我们的TRPM 3调节模型的预测，并阐明这种调节的分子决定因素。 使用分子生物学、膜片钳、平面脂质双层、皮肤神经电生理学 基于荧光的细胞成像技术和动物行为。

The topic of the original funded grant proposal was phosphoinositide regulation of the heat- and capsaicin sensitive TRPV1, and the cold- and menthol-sensitive TRPM8 channels. The current renewal proposal continues to study phosphoinositide regulation of TRPV1, and addresses an important unsolved problem, brought to light by a recent higher resolution structure in lipid nanodiscs, which found that the capsaicin/vanilloid binding site is occupied by a phosphoinositide, and proposed that it stabilizes the channel in the resting state, and vanilloids activate TRPV1 by replacing the lipid. PI(4,5)P2 and PI(4)P however are well established positive cofactors/regulators of TRPV1, which is difficult to reconcile with this model. The exact nature of the phosphoinositide lipid, however, is not well resolved in the structure. In Aim1 we will elucidate the nature of the phosphoinositide in the vanilloid binding site, using the combination of computational modeling, site directed mutagenesis, whole cell and excised patch electrophysiology and planar lipid bilayers. The TRPM3 ion channel is expressed in Dorsal Root Ganglion (DRG) neurons; its genetic deletion in mice results in altered sensitivity to noxious heat. TRPM3 is activated by heat, and chemical agonists, such as Pregnenolone Sulphate (PregS) and CIM0216. We found that this channel requires phosphoinositides for activity, and we also found that agonists of phospholipase C (PLC)-coupled receptors inhibit TRPM3. This inhibition, however, was not alleviated by intracellular delivery of excess PI(4,5)P2, and was reduced by a protein that binds the βγ subunits of heterotrimeric G-proteins (Gβγ sink). This finding points to the dominance of Gβγ signaling over PLC activation in regulating TRPM3. Activation of Gi-coupled receptors that do not activate PLC also robustly inhibited TRPM3 activity, and the effect was reduced by Gβγ sinks. Co-expression of Gβγ in intact cells, and application of purified Gβγ to excised inside-out patches also inhibited TRPM3, and we detected biochemical interaction between TRPM3 and Gβ by co-immunoprecipitation. These data suggest that Gβγ subunits are direct negative regulators of TRPM3. We also found that activation of endogenous Gi- coupled GABAB and opioid receptors inhibited PregS-induced Ca2+ signals in DRG neurons. In Aims 2 and 3, we will test predictions of our model of TRPM3 regulation, and elucidate the molecular determinants of this effect using a combination of molecular biology, patch clamp, planar lipid bilayer, skin-nerve electrophysiology, fluorescence-based cellular imaging techniques, and animal behavior.