Agonist-evoked changes in TRPV ion channel selectivity

辣椒素受体TRPV1是瞬时受体电位香草素的四个阳离子通道之一 (TRPV)亚家族，可在温暖或酷热的温度下激活。其中几个频道 (TRPV1、TRPV3、TRPV4)参与小鼠的热诱发痛觉。我们 最近发现，热诱导激活TRPV3会导致双相电流反应，反映出 初始通道被二价阳离子阻断，随后通过持续刺激移除该通道。这 数据块丢失取决于通道密度，一旦启动就会同步进行，并且是关联的 增加了对大分子有机阳离子N-甲基-d-葡萄糖胺(NMDG)的渗透性。动态离子 TRPV通道的选择性可能与神经递质释放、痛觉和 辣椒素的神经毒性，以及其他过程。因此，我们建议检验这些假设 TRPV3功能的变化反映了热诱导的通道孔的渐进性扩张，而TRPV1 在强烈的化学或热激活作用下也表现出气孔膨胀。此外，我们将确定 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， 分别进行了分析。使用电生理和荧光显微镜方法，我们将检查 TRPV1和TRPV3扩孔的机制基础及激动剂诱发的关系探讨 孔扩张和已知或推测的TRPV1和TRPV3调节机制，如 磷酸化和磷脂结合。最后，我们将寻求识别有选择性地 系统突变TRPV1和TRPV3对激动剂引起的TRPV1和TRPV3的扩孔很重要 在与选择性过滤器相邻的区域内。从这一筛选中产生的突变将被进一步研究。 因为它们有能力调节毛孔扩张的潜在“下游”效应，包括细胞的变化 自然和非自然细胞环境中的形态、细胞死亡和神经递质/细胞因子的释放。 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 并可为开发抗扩张拮抗剂或扩张药物提供合理的基础。 促进激动剂，用于治疗慢性疼痛。

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 isassociated 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.