Osmolyte Channels in Fish Erthrocytes:Functional and Molecular Properties

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Osmolyte Channels in Fish Erythrocytes: Functional and Molecular PropertiesLeon Goldstein Regulation of cell volume is an important physiological mechanism. Cells undergoing physiological dilution of the extracellular fluid and cell division, initially swell, and then, within minutes, restore their normal cell volume. This is accomplished by adjusting their intracellular solute concentrations to match that of the extracellular environment via the transport of osmotically active cell constituents (osmolytes) including ions and organic constituents. During cell swelling, specific protein channels are activated in the cell membrane, which lead to increased release of osmolytes from the swollen cells. Once cell volume is restored the channels are deactivated resulting in cessation of release of osmolytes from the previously swollen cells. Although the functional characteristics of the osmolyte channels have been detailed, their molecular identity is still not clear. Evidence suggests that the cell membrane protein, AE, is involved in the activation of osmolyte transport during cell swelling in fish red blood cells. Recently it has been discovered that there are three genes for the expression of AE proteins in skate (Raja erinacea) red blood cells. This research addresses the questions: Which genes are involved in expression of AE proteins in the red blood cell membranes? Which of the three proteins expressed in the cell membranes function as osmolyte transporters? What are the regulatory factors that activate the transporters during cell swelling? Intact skate red blood cells, isolated red blood cell membranes, and AE protein expression systems in frog eggs will be used to determine: 1. Which of the three AE proteins are expressed in the frog cell. 2. Which of the three AE proteins expressed in the frog egg cell membranes form osmolyte transport channels. 3.Which of the AE proteins in red blood cell membranes localize to lipid domains ('rafts') found on the surface of the cell membranes. 4. Which of several peripheral proteins found in the red blood cell membranes and attached to the AE protein are involved in the regulation of the cell membrane transporters. These studies will provide insights into the molecular and regulatory mechanisms involved in the control of cell volume. Undergraduate and graduate students will be recruited for the project from Brown University , the Mount Desert Island Biological Research Lab and from participation in an existing REU program. In addition, research experiences will be sponsored for high school students recruited from Providence and rural Maine.