Expression of TRPV1 and variant TRPV1 in the kidney

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（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中起重要作用。根据Northern分析和RNase保护试验，其他人假设存在仅在肾脏中表达的TRPV1的新剪接变体。到目前为止，还没有在任何组织中描述令人信服的剪接变异。在鉴定大鼠肾脏TRPV1克隆的过程中，我们发现了几个新的TRPV1剪接变体，预测它们编码具有小或大c端缺失的蛋白质。这些变异未在中枢或周围神经系统中发现。在其他模型中，包括相关的TRPC4通道，剪接变体编码了显性的负作用通道亚基。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，这种免疫反应性可能会因长期使用TRPV1激动剂辣椒素而下调。此外，MDCK细胞对TRPV1激动剂表现出强劲的钙瞬变。总的来说，这些数据表明肾小管细胞中存在一个完整的TRPV1系统，具有独特的调节特征（即剪接变异体）。

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.