Cell volume regulation is critical to overall cellular homeostasis and plays a role in a variety of pathological situations such as coma, edema, stroke and diabetes. This proposal uses a combination of electrophysiological, fluorescent and confocal microscopy and molecular biology techniques to address the physiological role of anions, osmolytes and the cytoskeleton in volume regulation. The focus is on a volume-regulated anion conductance (VRAC), which is a member of a ubiquitous family of mechanosensitive ion channels. This conductance is found in a wide variety of cell types and has been implicated in the recovery from cell swelling in many cells, including lymphocytes, the model system used in these studies. This proposal addresses several hypotheses directed toward understanding the regulation of this volume-regulated anion conductance and the contribution of this conductance to cellular volume regulation by concurrent measurements of current amplitude and cell swelling in a hyposmotic environment. Specifically addressed is the hypothesis that volume regulated anion current activity is regulated by the intracellular composition and concentration of osmolytes. Current and volume measurements will be combined with molecular biological techniques to address the hypothesis that the activity of this current is also regulated by a small, cytosolic actin binding protein, termed ICln. Changes in the actin cytoskeleton which provide cells with structure have also been implicated in regulating cell volume and have been suggested as the mechanotransducer which activated the anion current. This hypothesis will be addressed with two proteins, BotC2 and gelsolin to alter the equilibrium between F- and G- actin and determining the effect on current activation and cell swelling using electrophysiological, molecular and fluorescent techniques. Successful completion of this proposal will provide a greater understanding of the underlying mechanisms of mechanotransduction in cells and their role in volume regulation under normal and pathological conditions.
