Oscillatory Ca2 signaling in the C. elegans intestine

描述（由申请人提供）：细胞质Ca2+水平控制许多不同的细胞过程，包括基因表达、胞吐和分泌、运动和收缩、细胞增殖、程序性细胞死亡和分化。虽然生理学家对Ca2+信号事件有了深刻的了解，但许多基本问题仍未得到解答。线虫秀丽隐杆线虫为定义Ca2+信号的分子机制提供了许多实验优势。这些优势包括通过RNA干扰、敲除和转基因来操纵基因表达的相对容易和经济；现成的大量分子试剂和变异蠕虫菌株；一个完全测序和注释良好的基因组；以及进行诱变和向前遗传分析的能力。秀丽隐杆线虫的后体壁肌肉收缩（pBoc）驱动排便行为，并以每45-50秒有节奏的方式发生。基因分析已经确定了许多基因，当突变或敲除时，会破坏pBoc的节奏。这些包括编码IP3受体、PLC、K+通道和TRPM阳离子通道的基因。生理和分子研究表明，pBoc是由肠道上皮中有节奏的、依赖于ip的细胞内Ca2+振荡驱动的。最近，我们开发了原代秀丽隐杆线虫细胞培养方法，首次允许膜片钳表征肠细胞Ca2+电导。此外，我们已经开发了一种新的分离肠制备，允许细胞内Ca2+振荡的生理表征。我们将结合Ca2+成像、电生理学、反向遗传学和免疫荧光来验证PLC-p和PLC-y、KCNQ通道KQT-2和KQT-3以及TRPM通道GON-2和GTL-1共同调节细胞内Ca2+释放的假设。我们还将使用膜片钳电生理学和基因敲除来确定trpm样Ca2+通道ORCa是否由gon-2和/或gtl-1编码。我们将在研究中使用的实验方法的组合实际上更昂贵和耗时，或者在脊椎动物实验系统中不切实际。通过定义肠道Ca2+信号的基本方面，这一建议形成了一个长期努力的重要基础，将利用秀丽隐杆线虫的相当大的实验优势，以开发一个不可兴奋的细胞振荡Ca2+信号通路的综合分子理解。鉴于Ca2+信号的基本和高度保守的性质，从秀丽隐杆线虫获得的见解将清楚地为脊椎动物Ca2+信号传导机制提供新的和重要的见解。对Ca2+信号的详细分子理解对于理解和治疗包括癌症、心脏病和糖尿病在内的许多疾病过程至关重要。

DESCRIPTION (provided by applicant): Cytoplasmic Ca2+ levels control numerous, diverse cellular processes including gene expression, exocytosis and secretion, motility and contraction, cell proliferation, programmed cell death, and differentiation. While physiologists have gained an impressive understanding of Ca2+ signaling events, many fundamental questions remain unanswered. The nematode C. elegans provides numerous experimental advantages for defining molecular mechanisms of Ca2+ signaling. These advantages include relative ease and economy of manipulating gene expression by RNA interference, knockout and transgenesis; ready availability of numerous molecular reagents and mutant worm strains; a fully sequenced and well-annotated genome; and the ability to perform mutagenesis and forward genetic analysis. Posterior body wall muscle contraction (pBoc) in C. elegans drives defecation behavior and occurs in rhythmic fashion every 45-50 sec. Genetic analyses have identified numerous genes that, when mutated or knocked down, disrupt pBoc rhythm. These include genes encoding the IP3 receptor, PLC, K+ channels and TRPM cation channels. Physiological and molecular studies have demonstrated that pBoc is driven by rhythmic, IPs-dependent intracellular Ca2+ oscillations in the intestinal epithelium. Recently, we developed primary C. elegans cell culture methods that allow for the first time patch clamp characterization of intestinal cell Ca2+ conductances. In addition, we have developed a novel isolated intestine preparation that allows physiological characterization of intracellular Ca2+ oscillations. We will use a combination of Ca2+ imaging, electrophysiology, reverse genetics and immunofluorescence to test the hypothesis that PLC-p and PLC-y, the KCNQ channels KQT-2 and KQT-3, and the TRPM channels GON-2 and GTL-1 function together to regulate intracellular Ca2+ release. We will also use patch clamp electrophysiology and gene knockout to determine if the TRPM-like Ca2+ channel ORCa is encoded by gon-2 and/or gtl-1. The combination of experimental approaches we will use in our studies is substantially more costly and time-consuming, or not realistically possible in vertebrate experimental systems. By defining basic aspects of intestinal Ca2+ signaling, this proposal forms an essential foundation of a long-term effort that will exploit the considerable experimental advantages of C. elegans to develop an integrated molecular understanding of a non-excitable cell oscillatory Ca2+ signaling pathway. Given the fundamental and highly conserved nature of Ca2+ signaling, insights gained from C. elegans will clearly provide new and important insights into vertebrate Ca2+ signaling mechanisms. Detailed molecular understanding of Ca2+ signaling is essential for understanding and treating numerous disease processes including cancer, heart disease and diabetes.