Regulation Of Ca2+ Oscillations In Paramecium

描述（由申请人提供）：细胞内Ca+的振荡 在许多（如果不是所有的话）真核细胞中观察到浓度。他们一直 被认为支配着各种基本的生物过程，包括 分泌、增殖和发育。振荡通常是由 通过细胞外刺激，引起细胞内Ca+释放， 店细胞质Ca+浓度因此升高。存储耗尽是 然后向膜中的Ca+特异性电导发出信号，引发Ca+ 为准备随后的几波 release.尽管Ca+振荡的普遍性和重要性， 关于它们的起源和功能仍然有许多未解之谜， 包括存储耗尽信号的性质和它们被 调制的该提案调查了负责Ca+的途径 振荡，使用草履虫作为模型系统。这种单细胞使用Ca+ 振荡以影响对细胞外GTP的行为反应。草履虫是 特别适合这些研究，因为有证据表明， Ca+-特异性膜电导，响应于存储耗尽已经获得了 电压敏感性，使其特别容易引出和研究下， 电压箝位此外，可以获得不能响应GTP的突变体 用于振荡及其调节的遗传解剖 途径。该提案有三个具体目标。第一，电压钳技术 用于研究GTP应用后观察到的两种电流， 确定它们的性质和对电导的依赖性， 负责补充Ca+储备。第二个目标是调查 终止GTP持续暴露期间振荡的途径。 有证据表明，这涉及腺苷酸环化酶和蛋白激酶A。 该途径的目标被认为是储存-补充电流， 通过电压灵敏度的变化使其失活。最终目的 发现了新的突变体，这些突变体在感知和脱敏方面有缺陷， GTP这些突变体可以帮助解剖振荡的生理学， 它们的监管途径，并最终可以提供识别 参与这种Ca+信号传导现象的基因。

DESCRIPTION (provided by applicant): Oscillations in intracellular Ca+ concentration are observed in many, if not all eukaryotic cells. They have been suggested to govern a variety of fundamental biological processes, including secretion, proliferation and development. Oscillations are typically triggered by an extracellular stimulus that provokes Ca+ release from intracellular stores. Cytoplasmic Ca+ concentrations rise as a result. Store depletion is then signaled to a Ca+-specific conductance in the membrane, eliciting a Ca+ flux that replenishes the stores in preparation for subsequent waves of release. Despite the prevalence and importance of Ca+ oscillations, there remain many unanswered questions regarding their origins and function, including the nature of the store depletion signal and means by which they are modulated. This proposal investigates pathways responsible for Ca+ oscillations, using Paramecium as a model system. This unicell uses Ca+ oscillations to effect behavioral responses to extracellular GTP. Paramecium is particularly suited to these studies because evidence suggests that the Ca+-specific membrane conductance that responds to store depletion has gained a voltage sensitivity that makes it particularly easy to elicit and study under voltage clamp. Further, mutants that are unable to respond to GTP are available for use in a genetic dissection of the oscillations and their regulatory pathways. The proposal has three specific aims. First, voltage-clamp techniques are used to investigate two currents that are observed upon GTP application to determine their properties and dependence on the conductance that is believed to be responsible for replenishing the Ca+ stores. The second aim investigates the pathways that terminate the oscillations during persistent GTP exposure. Evidence suggests that this involves adenylate cyclase and protein kinase A. The target of this pathway is believed to be the store-replenishment current, inactivating it through a shift in voltage sensitivity. The final aim identifies new mutants that are defective in sensing and desensitization to GTP. These mutants can help dissect the physiology of the oscillations and their regulatory pathways and ultimately can provide the means to identify the genes involved in this Ca+ signaling phenomenon.