Transport of biogenic amines from the extracellular space to the cytoplasm and storage of these amines in intracellular secretory granules represent aspects of cellular physiology required for hemostasis, neurotransmission, and regulation of blood pressure and heart rate. These processes may be also involved in the response to antidepressant therapy. The granule membrane H+ pumping ATPase required for biogenic amine storage appears similar or identical to other intracellular ATPases responsible for such diverse cellular processes as receptor-mediated endocytosis, lysosomal acidification, and trans-epithelial H+ flux. To understand better the role of biogenic amine and ion transport, this proposal is directed toward understanding the molecular mechanisms of three transport systems: the plasma membrane serotonin transporter (which may also function as the receptor for the antidepressant imipramine), the reserpine-sensitive secretory granule amine transporter, and the granule H+ pumping ATPase. The molecular weights of each of transport component, solubilized in detergent, will be determined using a novel application of equilibrium sedimentation. The molecular form of serotonin transported by the granule amine transporter will be determined in membrane vesicles using fluorinated serotonin derivatives with altered pKa's. To ascertain whether the plasma membrane serotonin transporter is also the imipramine receptor, the Imipramine binding and serotonin transport reactions will be probed with a variety of antidepressants. The catalytic mechanism of ATP hydrolysis by the granule H+ pump will be studied using exchange ractions and stereochemically labeled ATP-S. Photoaffinity labels and suicide inhibitors will be used to label the enzyme's active site. Both the ATPase and the serotonin transporter will be purified from detergent solution. When sufficiently pure, the ATPase and transporter will be used to generate mono- and polyclonal antibodies which will, in turn, be used to identify and localize these proteins.
