Expression of TRPV1 and variant TRPV1 in the kidney

描述（由申请人提供）：TRPV 1是一种阳离子通道，最初克隆为辣椒素（或“辣椒”）受体。TRPV 1在外周神经系统中表达，在那里它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TRPV 1也在一些上皮细胞中表达（例如，结肠、肺和膀胱的那些），并且在感知包括壁张力和细胞酸中毒的有害应激源中起重要作用。基于北方分析和RNA酶保护测定，其他人已经假设存在仅在肾脏中表达的TRPV 1的新剪接变体。迄今为止，令人信服的剪接变异体尚未在任何组织中描述。在鉴定大鼠肾脏TRPV 1克隆的过程中，我们发现了几种新的TRPV 1剪接变体，预测其编码的蛋白质具有小的或大的C-末端缺失。这些变异在中枢和外周神经系统中均未发现。在其他模型中，包括相关的TRPC 4通道，剪接变体编码了占主导地位的负作用通道亚基。此外，我们注意到，从肾上皮MDCK细胞制备的裂解液中的抗TRPV 1免疫反应性，此外，这种免疫反应性可以下调长期治疗的TRPV 1激动剂，辣椒素。此外，MDCK细胞对TRPV 1激动剂的反应表现出强的钙瞬变。总之，这些数据表明肾小管细胞中的完整TRPV 1系统具有独特的调控特征（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、RNA酶保护和原位杂交技术的组合，鉴定TRPV 1和哺乳动物肾脏中新的肾脏特异性TRPV 1剪接变体的解剖学和亚细胞定位。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方法来描述变体TRPV 1在肾脏中表达的功能意义。长期目标是建立TRPV 1在肾脏中的感觉作用（通过解剖学和功能研究的结合），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TRPV 1功能的抑制剂或调节剂。此外，这样的试剂可能具有潜在的治疗效用关于这个重要的途径介导的伤害感受。

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.