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.