Divergent Calcium Channels of the Apicomplexan parasite Toxoplasma gondii

项目总结/摘要 Ca 2+离子几乎影响细胞生命的各个方面。Ca 2+信号传导始于Ca 2+通道的开放 在质膜（PM）或内质网（ER）中， 生理学上低（<100 nM）的胞质Ca 2+水平。Ca 2+浓度的时空变化 调节并使关键的生物过程，如基因的变化， 表达、细胞分化、肌肉收缩、受精或神经递质的分泌， 几个Ca 2+信号调节顶复门寄生虫如刚地弓形虫的致病途径， 感染了世界上大约三分之一的人口T.弓形虫是一种条件致病菌， 免疫功能低下的患者，如HIV感染者、胎儿和器官移植受者。作为 专性胞内病原体T.弓形虫在细胞内复制， 弓形虫病是其在细胞内生长和传播的直接结果。T.弓形虫依赖Ca 2+信号 对于刺激其感染周期的特定特征和几个Ca 2+信号传导元件起着至关重要的作用 寄生循环中的角色。然而，启动T.弓形虫主要是 可能是其生存能力和毒性所必需的。分子的发现和表征 启动T细胞中的Ca 2+信号。因此，弓形虫是了解其发病机制的核心。主动出口 中医从宿主细胞中分离出的弓形虫对于感染的传播是至关重要的，我们先前的工作提供了 结论性的证据表明，有一个胞质钙峰之前的出口。这种寄生性胞质增加 由细胞内储存物释放而产生，可能是内质网。然而，令人困惑的是， 细胞内寄生虫在低宿主胞质Ca 2+的包围下复制，但仍在其细胞内储存足够的Ca 2+。 急诊室启动出口。在宿主细胞破裂时，细胞外Ca 2+穿过PM内流有助于第二次细胞分裂。 Ca 2+峰值增强寄生虫的运动性，然后离开并寻找另一个宿主细胞入侵。我们的假设 PM Ca 2+内流对于细胞内Ca 2+库的再填充以及细胞内外Ca 2+来源是必不可少的 是触发导致寄生功能刺激的分子事件级联所必需的 如运动性、粘附素分泌、宿主细胞侵入、外出和传播。在本建议中，我们的目标是 表征使PM Ca 2+流入的蛋白质。几乎没有关于功能的信息。 钙通道的特性和作用。刚地。这种知识的缺乏可能是由于缺乏适当的 分子寄生虫学领域内的电生理学工具、技术和培训。我们解决这个 与一位哺乳动物电生理学家和一位建模师合作。渠道对于 寄生虫的成功单细胞生活，并且它们可以被许多治疗上有用的药剂靶向。离子 通道作为治疗靶点，甚至作为抗寄生虫剂，仍然明显未被充分利用。

PROJECT SUMMARY/ABSTRACT Ca2+ ions impact almost every aspect of cellular life. Ca2+ signaling begins with the opening of Ca2+ channels in either the plasma membrane (PM) or the endoplasmic reticulum (ER) and results in a dramatic increase in the physiologically low (<100 nM) cytosolic Ca2+ levels. The temporal and spatial Ca2+ levels are exquisitely regulated and enable the precise and specific activation of critical biological processes like changes in gene expression, cell differentiation, muscle contraction, fertilization, or secretion of neurotransmitters to name a few. Ca2+ signaling regulates pathogenic pathways of apicomplexan parasites like Toxoplasma gondii which infects approximately one third of the world’s population. T. gondii is an opportunistic pathogen of immunocompromised patients like HIV-infected individuals, fetuses, and organ transplant recipients. As an obligate intracellular pathogen, T. gondii replicates inside cells and the clinical manifestations of toxoplasmosis are a direct result of its growth within cells and its dissemination. T. gondii relies on Ca2+ signals for the stimulation of specific features of its infection cycle and several Ca2+ signaling elements play essential roles in its parasitic cycle. However, the fundamental elements that initiate Ca2+ signals in T. gondii are largely unknown yet are likely essential for its viability and virulence. Discovery and characterization of the molecules that initiate Ca2+ signaling in T. gondii are hence central for the understanding of its pathogenesis. Active egress of T. gondii from host cells is critical for dissemination of the infection and our prior work has provided conclusive evidence that there is a cytosolic Ca2+ peak preceding egress. This parasitic cytosolic increase arises from release from intracellular stores, likely the endoplasmic reticulum. It is puzzling, however, that intracellular parasites replicate surrounded by the low host cytosolic Ca2+ but still store sufficient Ca2+ in their ER to trigger egress. Upon host cell rupture, extracellular Ca2+ influx across the PM contributes to a second Ca2+ peak enhancing motility of parasites, which then exit and seek another host cell to invade. Our hypothesis is that PM Ca2+ entry is essential for refilling of intracellular Ca2+ stores, and both intra and extracellular sources are necessary for triggering the cascade of molecular events that lead to the stimulation of parasitic functions like motility, secretion of adhesins, invasion of host cells, egress and dissemination. In this proposal we aim to characterize the proteins that enable PM Ca2+ influx. There is almost no information about the functional characteristics and roles of Ca2+ channels in T. gondii. This lack of knowledge could be due to lack of appropriate tools, techniques, and training in electrophysiology within the molecular parasitology field. We address this void with a collaboration with a mammalian electrophysiologist and a modeler. Channels are critical for the successful unicellular life of parasites, and they could be targeted by many therapeutically useful agents. Ion channels remain significantly under-exploited as therapeutic targets, even more so as antiparasitic agents.