Agonist-evoked changes in TRPV ion channel selectivity

描述(申请人提供)：辣椒素受体，TRPV1，是瞬时受体潜在香草酸(TRPV)亚家族的四个阳离子通道之一，可在温暖或痛苦的高温下激活。这些通道中的几个(TRPV1、TRPV3、TRPV4)已被证明参与小鼠的热诱发痛觉。我们最近发现，热诱发的TRPV3激活导致双相电流响应，反映了二价阳离子最初的通道阻断，然后通过持续刺激消除该阻断。这种阻断的丧失依赖于通道密度，一旦启动，就会同步进行，并与大分子有机阳离子N-甲基-d-葡萄糖胺(NMDG)的渗透性增加有关。TRPV通道的动态离子选择性可能与神经递质释放、痛觉和辣椒素神经毒性等过程有关。因此，我们建议检验这样的假设，即TRPV3功能的这些变化反映了通道孔的渐进性热诱导扩张，并且TRPV1也表现出孔扩张对强烈的化学或热激活的响应。此外，我们将确定分别在感觉神经元和角质形成细胞中表达的天然TRPV1和TRPV3是否发生毛孔扩张。我们将利用电生理和荧光显微镜的方法，研究TRPV1和TRPV3的孔扩张的机制基础，并探索激动剂引起的孔扩张与已知或推测的TRPV1和TRPV3调节机制之间的关系，如磷酸化和磷脂结合。最后，我们将通过对选择性过滤器邻近区域内的残基进行系统的突变，寻找在TRPV1和TRPV3中对激动剂引起的孔扩张具有选择性重要的氨基酸。从这一筛选中产生的突变体将被进一步检测其调节潜在的毛孔扩张的“下游”效应的能力，包括在天然和非天然细胞环境中细胞形态、细胞死亡和神经递质/细胞因子释放的变化。这些研究将使我们能够确定TRPV1和TRPV3的离子选择性是否以及如何被调节，并可能为开发用于治疗慢性疼痛的抗扩张拮抗剂或促进扩张激动剂提供合理的基础。

DESCRIPTION (provided by applicant): The capsaicin receptor, TRPV1, is one of four cation channels of the transient receptor potential vanilloid (TRPV) subfamily that can be activated by warm or painfully hot temperatures. Several of these channels (TRPV1, TRPV3, TRPV4) have been shown to participate in heat-evoked pain sensation in mice. We recently found that heat-evoked activation of TRPV3 results in a biphasic current response that reflects an initial channel block by divalent cations and subsequent removal of that block by persistent stimulation. This loss of block is dependent on channel density, proceeds synchronously, once initiated, and is associated with increased permeability to the large organic cation N-methyl-d-glucamine (NMDG). Dynamic ion selectivity in TRPV channels could have implications for neurotransmitter release, pain sensation, and capsaicin neurbtoxicity, among other processes. We therefore propose to test the hypotheses that these changes in TRPV3 function reflect progressive heat-evoked dilation of the channel pore, and that TRPV1 also exhibits pore dilation in response to strong chemical or thermal activation. In addition, we will determine whether pore dilation occurs in native TRPV1 and TRPV3 expressed in sensory neurons and keratinocytes, respectively. Using electrophysiological and fluorescence microscopy approaches, we will examine the mechanistic basis of TRPV1 and TRPV3 pore dilation and explore the relationship between agonist-evoked pore dilation and known or presumed mechanisms of TRPV1 and TRPV3 regulation, such as phosphorylation and phospholipid binding. Finally, we will seek to identify amino acids that are selectively important for agonist-evoked pore dilation in TRPV1 and TRPV3 via systematic mutagenesis of residues within the domains adjacent to the selectivity filter. Mutants arising from this screen will be further examined for their abilities to mediate potential "downstream" effects of pore dilation, including changes in cell morphology, cell death, and neurotransmitter/cytokine release in native and non-native cellular contexts. These studies will allow us to determine whether and how the ion selectivities of TRPV1 and TRPV3 are regulated and may provide a rational basis for the development of either anti-dilation antagonists or dilation- promoting agonists, for the treatment of chronic pain.