Osmoregulation and Contractile Vacuole Function

[809929]即使细胞生活在恶劣的环境中，细胞也必须将其盐和水的含量维持在有利于生命的浓度。如果这些浓度明显偏离正常值，细胞就不能正常工作并死亡。这个项目的总体目标是发现生活在淡水中的单细胞原生动物草履虫是如何分泌水来维持水和盐的适当平衡的。这种原生动物有一个可收缩的液泡复合体，作为抽水细胞器排出水。然而，由于细胞器的膜是可自由渗透的，所以我们不知道细胞器是如何从细胞质的其余部分积累水分的。对这种矛盾的一种解释是，可收缩的液泡泵出并积聚盐分，使其液体内容物具有与细胞质相同的渗透压。水和盐一起被动地进入可收缩的液泡，两者一起被排出。然而，可能在草履虫中起这种作用的盐尚未被确定。为了测试这个模型并确定那些可能通过收缩液泡运输的盐，该项目将集中在以下具体目标上。(1)将离子选择电极插入生物体内可收缩的液泡中，以测定液泡液中的离子含量。(2)微电极将用于膜片钳夹细胞器，以研究离子电流，并识别可能存在于膜中的离子通道和泵。(3)将检验一个可以解释液泡中水积聚的具体假设。假设是质子梯度在液泡膜上形成，将碳酸氢盐离子作为反离子带入液泡。碳酸氢盐的积累会导致水被动地通过类似水通道的水通道流动。这项研究将为渗透调节（水盐平衡）如何从草履虫这样的“简单”生命形式进化到现在高等多细胞生物的状态提供线索。

9809929 Allen Cells must maintain their content of salts and water at concentrations that are favorable for life even though the cells live in a harsh environment. If these concentrations depart significantly from the norm, the cell cannot function properly and dies. The overall goal of this project is to discover how a single-cell protozoan, Paramecium, which lives in fresh water, is capable of secreting water to maintain this proper balance of water and salts. This protozoan has a contractile vacuole complex which acts as a pumping organelle to expel water. However, it is not known how the organelle accumulates water from the rest of the cytoplasm since the membrane of the organelle is freely permeable to water. One solution to this paradox would be that the contractile vacuole pumps and accumulates salts so that its fluid contents would have the same osmolarity as the cytoplasm. Water would enter the contractile vacuole passively with the salts and both would be expelled together. However, the salts that might serve this role in Paramecium have not been identified. To test this model and to identify those salts that might be transported by the contractile vacuole, this project will focus on the following specific aims. (1) Ion-selective electrodes will be inserted into the contractile vacuole in living organisms to determine ion content of the vacuole fluid. (2) Microelectrodes will be used to patch-clamp the organelle to study the ionic currents and to identify ion channels and pumps that may be present in the membrane. (3) A specific hypothesis will be tested that could account for water accumulation in the vacuole. The hypothesis is that a proton gradient develops across the vacuole membrane which brings bicarbonate ion into the vacuole as a counter ion. Accumulation of bicarbonate would then cause water to flow passively through aquaporin-like water channels. This study will provide clues on how osmoregulation (water and salt balance) has evolved from "simple" life forms such as Paramecium to its present state in higher multicellular organisms.