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

Pirt 和 Pirt2 的功能分析：TRP 通道的新型调节剂

• 批准号: 7782647
• 负责人: Xinzhong Dong
• 金额: $ 33.38万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7782647
• 关键词: Address; Affect; Afferent Neurons; Behavioral; Binding; Binding Proteins; Biochemical; Biological Process; C-terminal; Cells; Chemicals; Complex; Data; Esthesia; Family; Genes; Goals; In Vitro; Knockout Mice; Mediating; Menthol; Molecular; Mus; Names; Neuraxis; Neurons; Nociception; Pain; Pain management; Pharmaceutical Preparations; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Play; Property; Proteins; Regulation; Research; Role; Site; Spinal Ganglia; System; TRPV1 gene; Techniques; Testing; Transmembrane Domain; Whole-Cell Recordings; Wild Type Mouse; Xenopus oocyte; behavior test; chronic pain; cold temperature; in vivo; novel; public health relevance; receptor; response; sensor

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.

描述（由申请人提供）：我们研究的长期目标是了解如何调节TRP活动。最近，我们鉴定出一种新型基因，称为TRP（PIRT）的磷酸肌醇相互作用调节剂，该调节剂在背部根神经节（DRG）中高度表达，或在中枢神经系统中不存在伤害性神经元。 PIRT编码两个跨膜结构域蛋白。 PIRT NULL小鼠的行为和电生理研究表明，PIRT是TRPV1的阳性调节剂。生化分析表明，PIRT结合TRP和磷脂酰肌醇-4,5-双磷酸（PIP2）。重要的是，PIRT通过PIP2增强了TRPV1活性。我们的初步数据表明，PIRT还可以结合和调节TRPM8，这是一种用于冷感觉和薄荷醇的分子传感器。此外，我们确定了另一种与结构相关的PIP2结合蛋白，称为PIRT2。有趣的是，PIRT2还结合了包括TRPA1和TRPV1在内的某些TRP。在此提案中，我们将采用行为，电生理和生化方法进一步研究PIRT和PIRT2在调节这些PIP2敏感TRP通道中的作用。目的I是通过比较野生型和pirt null小鼠的神经元在培养的DRG神经元中发现PIRT对TRPM8的积极作用。我们将研究PIRT如何影响单个通道级别TRPM8的通道性质。为了确定PIRT在体内是否影响TRPM8介导的冷感觉，我们将在PIRT NULL小鼠及其野生型同窝仔的行为测试中进行先前描述的行为测试。在AIM II中，我们将研究PIRT调节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中哪些结构域才需要其结合。然后，我们将评估PIRT和PIP2是否需要彼此增强TRPM8。 AIM III是确定PIRT2是否在通过PIP2的TRPA1调制中起作用。我们将检验以下假设：PIRT2参与PIP2对TRPV1的抑制作用。通过分析PIRT2基因敲除小鼠的行为表型，我们将知道PIRT2是否在TRPA1-和TRPV1介导的疼痛感觉中起作用。提出的对PIRT和PIRT2的分析将有助于我们对TRP调节的理解。 公共卫生相关性：瞬态受体电位（TRP）渠道参与许多基本生物学过程，包括疼痛感。我们的研究表明，PIRT和PIRT2代表了TRP通道调节剂的新型家族。因此，PIRT和PIRT2的功能分析应促进我们对TRP调制的理解，从而为开发新的药物打开门以治疗诸如慢性疼痛之类的疾病。