Functional Analysis of Pirt and Pirt2: novel regulators of TRP channels

描述（由申请人提供）：我们研究的长期目标是了解TRP活动是如何调节的。最近，我们发现了一个新的基因，称为磷酸肌醇相互作用调节TRP（Pirt），这是高度表达的疼痛感觉，或伤害性神经元在背根神经节（DRG）和缺乏中枢神经系统。Pirt编码一个双跨膜结构域蛋白。Pirt基因敲除小鼠的行为和电生理学研究表明，Pirt作为TRPV 1的正调节剂发挥作用。生化分析表明，Pirt结合TRP和磷脂酰肌醇-4，5-二磷酸（PIP 2）。重要的是，Pirt通过PIP 2增强TRPV 1活性。我们的初步数据表明，Pirt也可以结合和调节TRPM 8，一种冷感觉和薄荷醇的分子传感器。此外，我们鉴定了另一种结构上相关的PIP 2结合蛋白，称为Pirt 2。有趣的是，Pirt 2也结合某些TRP，包括TRPA 1和TRPV 1。在这个提议中，我们将采取行为，电生理和生物化学的方法来进一步研究Pirt和Pirt 2在调节这些PIP 2敏感的TRP通道中的作用。目的I是通过比较野生型和Pirt缺失小鼠的神经元来评估Pirt对TRPM 8的积极作用是否也在培养的DRG神经元中发现。我们将研究Pirt如何在单通道级别影响TRPM 8的通道属性。为了确定Pirt是否在体内影响TRPM 8介导的冷感觉，我们将对Pirt缺失小鼠及其野生型同窝仔进行先前描述的行为测试。在目标II中，我们将研究Pirt如何调节TRPM 8的分子机制。我们将采用生物化学方法来确定TRPM 8和Pirt是否在DRG神经元中形成复合物，以及Pirt和TRPM 8中的哪些结构域是它们结合所必需的。然后，我们将评估Pirt和PIP 2是否需要彼此来增强TRPM 8。目的III是确定Pirt 2是否通过PIP 2在TRPA 1的调节中起作用。我们将检验Pirt 2参与PIP 2对TRPV 1的抑制作用的假设。通过分析Pirt 2基因敲除小鼠的行为表型，我们将了解Pirt 2是否在TRPA 1和TRPV 1介导的疼痛感觉中起作用。Pirt和Pirt 2的拟议分析将有助于我们理解TRP监管。 公共卫生相关性：瞬时受体电位（TRP）通道参与许多基本的生物过程，包括痛觉。我们的研究表明，Pirt和Pirt 2代表了一个新的家庭的TRP通道调节剂。因此，Pirt和Pirt 2的功能分析应该有助于我们理解TRP调节，从而为开发治疗慢性疼痛等疾病的新药打开大门。

DESCRIPTION (provided by applicant): The long-term objective of our research is to understand how TRP activities are regulated. Recently, we identified a novel gene, called phosphoinositide interacting regulator of TRP (Pirt), which is highly expressed in pain-sensing, or nociceptive neurons in dorsal root ganglia (DRG) and absent in the central nervous system. Pirt encodes a two-transmembrane domain protein. Behavioral and electrophysiological studies of Pirt null mice suggest that Pirt functions as a positive regulator of TRPV1. Biochemical analysis shows that Pirt binds both TRPs and phosphatidylinositol-4,5-bisphosphate (PIP2). Importantly, Pirt enhances TRPV1 activity via PIP2. Our preliminary data suggest that Pirt can also bind and modulate TRPM8, a molecular sensor for cold sensation and menthol. In addition, we identified another structurally related PIP2 binding protein, called Pirt2. Interestingly, Pirt2 also binds certain TRPs including TRPA1 and TRPV1. In this proposal, we will take behavioral, electrophysiological, and biochemical approaches to further study the role of Pirt and Pirt2 in regulating these PIP2-sensitive TRP channels. Aim I is to assess whether the positive effect of Pirt on TRPM8 is also found in cultured DRG neurons by comparing neurons from wild-type and Pirt null mice. We will study how Pirt affects channel properties of TRPM8 at the single channel level. To determine whether Pirt affects TRPM8-mediated cold sensation in vivo, we will perform previously described behavioral tests on Pirt null mice and their wild-type littermates. In Aim II, we will study the molecular mechanism of how Pirt regulates TRPM8. We will employ biochemical approach to determine whether TRPM8 and Pirt form a complex in DRG neurons and which domains in Pirt and TRPM8 are required for their binding. Then we will assess whether Pirt and PIP2 require each other to enhance TRPM8. Aim III is to determine whether Pirt2 plays role in modulation of TRPA1 via PIP2. We will test the hypothesis that Pirt2 is involved in the PIP2 inhibitory effect on TRPV1. By analyzing the behavioral phenotypes of Pirt2 knockout mice, we will know whether Pirt2 plays role in TRPA1- and TRPV1-mediated pain sensation. The proposed analysis of Pirt and Pirt2 will facilitate our understanding of TRP regulation. PUBLIC HEALTH RELEVANCE: Transient receptor potential (TRP) channels are involved in many fundamental biological processes including pain sensation. Our studies suggest that Pirt and Pirt2 represent a novel family of TRP channel regulators. Therefore, functional analysis of Pirt and Pirt2 should facilitate our understanding of TRP modulation and thereby open the door for developing novel drugs to treat conditions like chronic pain.