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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL021217-10
Application #
3336424
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Humphreys, C J; Wall, S C; Rudnick, G (1994) Ligand binding to the serotonin transporter: equilibria, kinetics, and ion dependence. Biochemistry 33:9118-25
Rudnick, G; Steiner-Mordoch, S S; Fishkes, H et al. (1990) Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter. Biochemistry 29:603-8
Waldman, B C; Rudnick, G (1990) UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP. Biochemistry 29:44-52
Waldman, B C; Rudnick, G (1989) A method for replacing intravesicular contents of Golgi vesicles using an air-driven ultracentrifuge. Anal Biochem 180:216-21
Rudnick, G; Kirk, K L; Fishkes, H et al. (1989) Zwitterionic and anionic forms of a serotonin analog as transport substrates. J Biol Chem 264:14865-8
Humphreys, C J; Cassel, D; Rudnick, G (1989) 2-Iodoimipramine, a novel ligand for the serotonin transporter. Mol Pharmacol 36:620-6
Humphreys, C J; Levin, J; Rudnick, G (1988) Antidepressant binding to the porcine and human platelet serotonin transporters. Mol Pharmacol 33:657-63
Dean, G E; Nelson, P J; Agnew, W S et al. (1987) Hydrodynamic properties of the chromaffin granule hydrogen ion pumping adenosinetriphosphatase. Biochemistry 26:2301-5
Dean, G E; Nelson, P J; Rudnick, G (1986) Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules. Biochemistry 25:4918-25
Arvan, P; Rudnick, G; Castle, J D (1985) Relative lack of ATP-driven H+ translocase activity in isolated parotid secretory granules. J Biol Chem 260:14945-52