Expression of TRPV1 and variant TRPV1 in the kidney

TRPV1 和变异体 TRPV1 在肾脏中的表达

• 批准号: 6759776
• 负责人: DAVID M COHEN
• 金额: $ 12.6万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6759776
• 关键词: MDCK cell; RNase protection assay; Xenopus; biological polymorphism; calcium indicator; cations; cell line; confocal scanning microscopy; electrophysiology; immunocytochemistry; in situ hybridization; kidney; laboratory rat; membrane channels; protein protein interaction; reagent /indicator; receptor expression; urinary bladder epithelium; western blottings

DESCRIPTION (provided by applicant): TRPV1 is a cation channel that was originally cloned as the capsaicin (or "hot pepper") receptor. TRPV1 is expressed in the peripheral nervous system where it serves to sense and integrate a large number of noxious stimuli such as heat, protons, and lipid mediators. Very recent data indicate that TRPV1 is also expressed in some epithelial cells (e.g., those of the colon, lung, and bladder) and plays a vital role in sensing noxious stressors including wall tension and cellular acidosis. Based upon Northern analysis and RNase protection assays, others have postulated the existence of novel splice variants of TRPV1 expressed only in the kidney. To date, convincing splice variants have not been described in any tissue. In the course of identifying a rat kidney TRPV1 clone, we discovered several novel splice variants of TRPV1 predicted to encode proteins with either small or large C-terminal deletions. These variants were not found in central or peripheral nervous system. In other models, including the related TRPC4 channel, splice variants have encoded dominant negative-acting channel subunits. In addition, we noted anti-TRPV1 immunoreactivity in lysates prepared from renal epithelial MDCK cells; furthermore, this immunoreactivity could be downregulated by protracted treatment with the TRPV1 agonist, capsaicin. Moreover, MDCK cells exhibited robust calcium transients in response to TRPV1 agonists. In aggregate, these data suggested an intact TRPV1 system in kidney tubule cells with unique regulatory features (i.e., splice variants). As such, the first objective is to identify the anatomic and subcellular localization of TRPV1 and of the novel kidney-specific TRPV1 splice variants in the mammalian kidney, through a combination of immunoblotting, immunohistochemistry, RNase protection, and in situ hybridization techniques. The second objective is to describe the functional significance of variant TRPV1 expression in the kidney, via fura-2-based calcium imaging, protein interaction strategies, and electrophysiological methods. The long-term objectives are to establish the sensory role of TRPV1 in the kidney (through a combination of anatomic and functional studies), and to determine whether one of these unique kidney-specific splice products encodes either a channel with unique properties, or an inhibitor or modulator of TRPV1 function. In addition, such a reagent might have potential therapeutic utility with respect to this important pathway mediating nociception.

描述（由申请人提供）：TRPV1 是一种阳离子通道，最初被克隆为辣椒素（或“辣椒”）受体。 TRPV1 在周围神经系统中表达，用于感知和整合大量有害刺激，如热、质子和脂质介质。最近的数据表明，TRPV1 也在一些上皮细胞（例如结肠、肺和膀胱的上皮细胞）中表达，并且在感知有害应激源（包括壁张力和细胞酸中毒）中发挥着至关重要的作用。基于 Northern 分析和 RNase 保护测定，其他人推测存在仅在肾脏中表达的 TRPV1 的新剪接变体。迄今为止，尚未在任何组织中描述令人信服的剪接变体。在鉴定大鼠肾 TRPV1 克隆的过程中，我们发现了 TRPV1 的几种新剪接变体，预计编码具有小或大 C 端缺失的蛋白质。在中枢或周围神经系统中未发现这些变异。在其他模型中，包括相关的 TRPC4 通道，剪接变体编码了主要的负作用通道亚基。此外，我们注意到肾上皮 MDCK 细胞制备的裂解物具有抗 TRPV1 免疫反应性；此外，这种免疫反应性可以通过TRPV1激动剂辣椒素的长期治疗来下调。此外，MDCK 细胞对 TRPV1 激动剂表现出强烈的钙瞬变反应。总的来说，这些数据表明肾小管细胞中存在完整的 TRPV1 系统，具有独特的调节特征（即剪接变体）。 因此，第一个目标是通过免疫印迹、免疫组织化学、RNase 保护和原位杂交技术的组合来鉴定 TRPV1 和新型肾脏特异性 TRPV1 剪接变体在哺乳动物肾脏中的解剖和亚细胞定位。第二个目标是通过基于 fura-2 的钙成像、蛋白质相互作用策略和电生理学方法来描述肾脏中 TRPV1 变异表达的功能意义。长期目标是确定 TRPV1 在肾脏中的感觉作用（通过解剖学和功能研究相结合），并确定这些独特的肾脏特异性剪接产物之一是否编码具有独特特性的通道，或者编码具有独特性质的通道。 TRPV1 功能的抑制剂或调节剂。此外，这种试剂对于介导伤害感受的这一重要途径可能具有潜在的治疗效用。