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活动是如何被调节的。最近，我们发现了一个新的基因，称为磷酸肌苷相互作用调节剂TRP (Pirt)，它在背根神经节（DRG）的痛觉或伤害性神经元中高度表达，而在中枢神经系统中缺失。Pirt编码一种双跨膜结构域蛋白。Pirt缺失小鼠的行为和电生理研究表明，Pirt是TRPV1的正调节因子。生化分析表明，Pirt能结合TRPs和磷脂酰肌醇-4,5-二磷酸（PIP2）。重要的是，Pirt通过PIP2增强了TRPV1的活性。我们的初步数据表明，Pirt还可以结合并调节TRPM8， TRPM8是一种冷感和薄荷醇的分子传感器。此外，我们还鉴定了另一种结构相关的PIP2结合蛋白，称为Pirt2。有趣的是，Pirt2还结合某些TRPs，包括TRPA1和TRPV1。在本课题中，我们将采用行为学、电生理和生化等方法进一步研究Pirt和Pirt2在调节这些pip2敏感的TRP通道中的作用。目的一是通过比较野生型和无Pirt小鼠的神经元，评估Pirt对TRPM8的积极作用是否也存在于培养的DRG神经元中。我们将在单通道水平上研究ptt如何影响TRPM8的通道特性。为了确定Pirt是否会影响体内trpm8介导的冷感觉，我们将对Pirt缺失小鼠及其野生型幼崽进行先前描述的行为测试。在Aim II中，我们将研究Pirt调控TRPM8的分子机制。我们将采用生化方法来确定TRPM8和Pirt是否在DRG神经元中形成复合物，以及Pirt和TRPM8的哪些结构域是它们结合所必需的。然后我们将评估Pirt和PIP2是否需要彼此增强TRPM8。目的III是确定Pirt2是否通过PIP2调节TRPA1。我们将检验Pirt2参与PIP2对TRPV1抑制作用的假设。通过分析Pirt2敲除小鼠的行为表型，我们将了解Pirt2是否在TRPA1-和trpv1介导的疼痛感觉中发挥作用。对Pirt和Pirt2的分析将有助于我们对TRP调控的理解。

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.