Agonist-evoked changes in TRPV ion channel selectivity

辣椒素受体TRPV 1是瞬时受体电位香草酸的四个阳离子通道之一 （TRPV）亚家族，其可被温暖或令人痛苦的高温激活。其中一些渠道 TRPV 1、TRPV 3、TRPV 4参与小鼠热诱发的痛觉。我们 最近发现，热诱发的TRPV 3激活导致双相电流反应，反映了 最初通道被二价阳离子阻断，随后通过持续刺激去除阻断。这 数据块丢失取决于通道密度，一旦启动，同步进行，并且关联 对大有机阳离子N-甲基-D-葡糖胺（NMDG）的渗透性增加。动态离子 TRPV通道的选择性可能对神经递质释放、疼痛感觉和 辣椒素神经毒性等。因此，我们建议测试这些假设， TRPV 3功能的变化反映了通道孔的进行性热诱发扩张，TRPV 1 也表现出响应于强化学或热活化的孔扩张。此外，我们将确定 孔扩张是否发生在感觉神经元和角质形成细胞中表达的天然TRPV 1和TRPV 3中， 分别使用电生理学和荧光显微镜方法，我们将研究 TRPV 1和TRPV 3孔扩张的机制基础，并探讨激动剂诱发的 孔扩张和已知或推测的TRPV 1和TRPV 3调节机制，如 磷酸化和磷脂结合。最后，我们将寻求确定选择性地被 通过残基系统性诱变对TRPV 1和TRPV 3中激动剂诱发的孔扩张是重要的 在与选择性滤波器相邻的域内。将进一步检查筛选出的突变体 因为它们能够介导孔扩张的潜在“下游”效应，包括细胞内的变化， 形态学、细胞死亡和天然和非天然细胞环境中的神经递质/细胞因子释放。 这些研究将使我们能够确定TRPV 1和TRPV 3的离子选择性是否以及如何被改变。 调节，并可能提供一个合理的基础，无论是抗扩张拮抗剂或扩张- 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.