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. ? ?
