Regulation of sensory TRP channels by phospholipids and G-proteins

最初拨款提案的主题是肌醇磷脂对辣椒素和辣椒素的调节。 敏感的TRPV1，以及对寒冷和薄荷醇敏感的TRPM8通道。目前的续期提案 继续研究肌醇磷脂对TRPV1的调节，并解决了一个重要的悬而未决的问题， 最近在脂质纳米盘中发现了一种更高分辨率的结构，它发现 辣椒素/香草素结合部位被一种磷脂酰肌醇占据，并认为它能稳定通道。 静息状态，香草素通过取代脂质来激活TRPV1。PI(4，5)P2和PI(4)P表现良好 建立了TRPV1的正协因子/调节因子，这与这个模型很难调和。完全相同的 然而，肌醇磷脂的性质在结构中并没有得到很好的分解。在Aim1中，我们将阐明 香草素结合部位中的磷脂酰肌醇的性质，使用计算模型的组合， 定点突变，全细胞和切除的斑块电生理和平面脂质双分子层。这个 TRPM3离子通道在小鼠背根神经节(DRG)神经元中表达；其基因缺失导致 对有毒高温的敏感度发生了变化。TRPM3被加热激活，而化学激动剂，如 孕烯醇酮硫酸盐(PregS)和CIM0216。我们发现这个通道需要磷脂酰肌醇 活性，我们还发现磷脂酶C(PLC)偶联受体的激动剂抑制TRPM3。这 然而，细胞内传递过量的PI(4，5)P2并不能减轻抑制作用，而是通过 与异源三聚体G蛋白的βγ亚基结合的蛋白质(Gβγ汇)。这一发现指向了优势 G-βγ信号通过激活PLC来调节TRPM3。激活Gi偶联受体而不是 激活PLC也能显著抑制TRPM3的活性，这种抑制作用可被G-βγ受体所减弱。共同表达 在完整细胞中Gβγ的表达，以及将纯化的Gβγ应用于切除的内向外贴片也抑制了TRPM3，以及 用免疫共沉淀法检测TRPM3与G-β之间的生化相互作用。这些数据表明 Gβγ亚基是TRPM3的直接负调控因子。我们还发现，内源性GI-1的激活 GABAB和阿片受体偶联可抑制PregS诱导的DRG神经元钙信号。在目标2和目标3中， 我们将测试我们的TRPM3调控模型的预测，并阐明其分子决定因素。 结合分子生物学、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.