Regulation of GTP-Induced Depolarizations in Paramecium

小行星9410756 草履虫是研究真核细胞中感觉传导机制的一个极好的系统，因为可以选择行为突变体来遗传解剖感觉传导途径。 此外，游泳行为可以用作一种方便的生物测定，以估计药物，抗体和突变对细胞电生理特性的影响。 在草履虫中，引诱剂通常引起超极化和快速向前游泳，而驱避剂则会引起向后游泳。 外部GTP在低至0.01微摩尔的浓度下是驱避剂。 它激发了新的重复性躯体（身体）去极化，导致向后游泳的发作。 1分钟内发生适应，导致恢复正常游泳。 本研究的目的是验证这一假设，即外部GTP被识别的身体质膜嘌呤受体激活一种新的体细胞钙电导。 这触发了重复的躯体平台去极化。 如果涉及受体操纵的通道，则可能存在第二信使参与它们的激活、失活或适应。 其目标是：1）鉴定和表征负责GTP诱导的体细胞去极化的离子通道或转运蛋白，并描述离子依赖性、药理学、动力学和可能的第二信使参与。 将使用已建立的细胞内电流注入和电压钳程序。 在暴露于GTP期间，将测定第二信使cAMP、cGMP和钙; 2）使用行为生物测定来筛选药物和突变对对外部GTP的反应的影响。 新的行为突变体将通过行为选择技术进行选择和表征;和3）使用行为突变体的显微注射"固化"来鉴定突变体的改变的基因产物。 目的是了解草履虫在GTP诱导的反应和感觉适应中所涉及的生化和电生理机制。 这将有助于更好地了解真核细胞的嘌呤能反应和化学感受性转导机制。 %活细胞可以感知并对环境中的化学物质做出反应。 这种细胞反应的第一个迹象通常是细胞膜电特性的变化。这项研究的目的是了解单细胞如何感知化学物质并对其做出反应。 草履虫这种自由游动的单细胞生物为研究这些反应的分子基础提供了极好的机会。 草履虫可以在类似于池塘水的溶液中大量生长;其膜的电特性可以使用电生理学技术进行研究;其遗传学已经很好地理解，并且可以产生影响生物体游泳行为的突变体。 游泳行为可以作为一种方便的测定其化学传感机制的变化。 草履虫在很低的浓度下就能很好地感受到一种化学物质，它是一种有机分子，GTP。 外部GTP对这些细胞是一种强大的排斥剂。 它们在低至10.0纳摩尔的浓度下游泳离开。 许多类型的行为突变体将产生不响应GTP，其生化和生理缺陷的细节将被表征。 通过这种方式，将确定细胞GTP感觉通路的重要组成部分。 这项研究的结果将为细胞如何感知环境中的化学物质并对其做出反应提供重要的见解。 ***

9410756 Hennessey Paramecium is an excellent system for studying sensory transduction mechanisms in eukaryotic cells because behavioral mutants can be selected to genetically dissect a sensory transduction pathway. Also, swimming behavior can be used as a convenient bioassay to estimate the effects of drugs, antibodies, and mutations on the electrophysiological properties of the cell. In Paramecium, attractants generally cause hyperpolarization and fast forward swimming while repellents depolarize and can cause backward swimming. External GTP is a repellent at concentrations as low as 0.01 micromolar. It elicits novel repetitive somatic (body) depolarizations which cause bouts of backward swimming. Adaptation occurs within 1 minute, causing a return to normal swimming. This research is designed to test the hypothesis that external GTP is recognized by a body plasma membrane purinergic receptor to activate a novel somatic calcium conductance. This triggers repetitive somatic plateau depolarizations. if receptor-operated channels are involved, there may be second messenger involvement in either their activation, deactivation, or adaptation. The objectives are to: 1) Identify and characterize the ion channels or transporters responsible for GTP-induce somatic depolarizations and describe the ion dependence, pharmacology, kinetics, and possible second messenger involvements. Established intracellular current injection and voltage clamp procedures will be used. The second messengers cAMP, cGMP and calcium will be assayed during exposure to GTP; 2) Use behavioral bioassays to screen drugs and mutations for effects on responses to external GTP. New behavioral mutants will be selected by behavioral selection techniques and characterized; and 3) Use microinjection "curing" of behavioral mutants to identify the altered gene products of mutants. The goal is to understand the biochemical and electrophysiological mechanisms involved in GTP-induced responses and sensory a daptation in Paramecium. This will contribute to a better understanding of purinergic responses and chemosensory transduction mechanisms in eukaryotic cells. %%% Living cells can sense and respond to chemicals in their environment. The first indication of such a cellular response is usually a change in the electrical properties of the cell membrane. The goal of this research is to understand how single cells sense chemicals and respond to them. The free-swimming, single-celled organism, Paramecium provides an excellent opportunity to study the molecular bases of these responses. Paramecium can be grown in large numbers in solutions resembling pond water; the electrical properties of its membrane can be studied using techniques of electrophysiology; and its genetics are well understood and mutants can be generated that affect the organism's swimming behavior. Swimming behavior can be used as a convenient assay for changes in its chemical sensing machinery. One chemical that Paramecium can sense very well at very low concentrations is an organic molecule, GTP. External GTP is a powerful repellent for these cells. They swim away from it at concentrations as low as 10.0 nanomolar. Many types of behavioral mutants will be generated that do not respond to GTP, and the details of their biochemical and physiological defects will be characterized. In this way the important components of the cellular GTP sensation pathway will be identified. The results of this research will provide important insights into how cells sense chemicals in their environment and respond to them. ***