Expression of TRPV1 and variant TRPV1 in the kidney

说明(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在外周神经系统中表达，在那里它负责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(如结肠、肺和膀胱的上皮细胞)中表达，并在感知包括壁张力和细胞酸中毒在内的有害应激源方面发挥重要作用。基于Northern分析和核糖核酸酶保护分析，其他人推测存在仅在肾脏表达的TRPV1的新剪接变体。到目前为止，还没有在任何组织中描述过令人信服的剪接变体。在鉴定大鼠肾脏TRPV1克隆的过程中，我们发现了几个新的TRPV1剪接变异体，被预测为编码具有小的或大的C-末端缺失的蛋白质。这些变异在中枢或外周神经系统中均未发现。在其他模型中，包括相关的TRPC4通道，剪接变体编码了主导的负作用通道亚基。此外，我们注意到从肾上皮MDCK细胞制备的裂解物中有抗TRPV1的免疫反应；此外，这种免疫反应可以被TRPV1激动剂辣椒素长期治疗而下调。此外，MDCK细胞对TRPV1激动剂有强烈的钙瞬变反应。总而言之，这些数据表明，在肾小管细胞中存在完整的TRPV1系统，具有独特的调节功能(即剪接变体)。 因此，第一个目标是通过结合免疫印迹、免疫组织化学、核糖核酸酶保护和原位杂交技术，鉴定TRPV1和新的肾脏特异性TRPV1剪接变异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方法，描述不同TRPV1在肾脏中表达的功能意义。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中是否有一种编码具有独特属性的通道，或者是TRPV1功能的抑制或调节因子。此外，这种试剂可能对介导伤害性感觉的这一重要途径具有潜在的治疗作用。

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.