In the last decade, it has become apparent that most secretory vesicles have an inwardly directed proton-translocating ATPase. Because this proton pump generates a membrane potential (inside positive) and because biological membranes typically have a small but perceptible Cl- conductance, secretory vesicles tend to accumulate chloride. This poses a potential threat to the osmotic stability of the vesicles. We propose that Cl- is eliminated from secretory vesicles and osmotic balance is maintained by a chloride/bicarbonate exchange. Because secretory vesicles have acidic interiors, they also have a relatively low internal HC03- concentration. Internal HC03- tends to become protonated and to diffuse out of the organelles as H2C03- Therefore, the pH gradient across secretory vesicle membranes (acidic inside) should set up a bicarbonate concentration gradient (high outside) which in turn should drive anion exchange expelling Cl- in exchange for HC03-. This hypothesis will be tested and the proposed Cl-/HC03- exchange characterized using bovine chromaffin vesicles, the catecholamine storage vesicles of the adrenal medulla. Specific objectives are 1) to develop assays for anion exchange activity and characterize inhibitors, 2) to characterize the kinetics of anion exchange for comparison with the """"""""band 3"""""""" anion exchange protein in bovine erythrocytes, 3) to characterize the structure of the anion exchange protein for comparison with the red blood cell anion exchange protein, and 4) to evaluate, using cultured chromaffin cells, the importance of anion exchange in secretory vesicle biogenesis, stability and exocytosis in vivo. At present, nothing is known about osmotic stability and volume regulation in organelles. These processes could be essential for such vital cellular functions as secretory vesicle formation and secretion itself. Cellular dysfunctions associated with defects in organelles anion exchange will undoubtedly follow from an understanding of this phenomenon in normal cells.
