Regulation Of Ca2+ Oscillations In Paramecium

描述(申请人提供)：细胞内钙离子振荡 在许多真核细胞中观察到浓度，如果不是所有真核细胞的话。他们一直都是 建议管理各种基本的生物过程，包括 分泌、增殖和发育。振荡通常会被触发 通过细胞外刺激刺激细胞内钙离子释放 商店。胞质内钙离子浓度也随之升高。门店耗尽是 然后向细胞膜中的钙离子特定电导发出信号，从而引发钙离子 补充存储以准备后续浪潮的流量 放手。尽管钙离子振荡很普遍，也很重要，但 关于它们的起源和功能仍有许多悬而未决的问题， 包括存储耗尽信号的性质和它们被 调制过的。这项建议调查了负责钙离子的途径。 振荡，以草履虫为模型系统。这种单细胞使用Ca+ 影响细胞外GTP行为反应的振荡。草履虫是 特别适合于这些研究，因为有证据表明 响应存储耗尽的特定于Ca+的膜电导增加了 电压敏感度，使其特别容易在 电压钳位。此外，还可以获得不能对GTP作出反应的突变体 用于对振荡及其调控的遗传解剖 小路。该提案有三个具体目标。第一，电压钳位技术 用于研究在GTP应用于 确定它们的性质和对所认为的电导的依赖 负责补充Ca+储备库。第二个目的是调查 在持续的GTP暴露过程中终止振荡的途径。 有证据表明，这与腺苷环化酶和蛋白激酶A有关。 该通路的目标被认为是存储-补充电流， 通过电压敏感度的变化来使其失活。最终目标 识别在感知和脱敏方面有缺陷的新突变体 GTP。这些突变体可以帮助剖析振荡的生理学和 它们的调控途径，并最终可以提供识别 参与这一钙信号现象的基因。

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.