This project will elucidate the biochemical mechanisms responsible for storage of the neurotransmitter acetylcholine (ACh) by synaptic vesicles of nerve terminals. The guiding hypothesis is that recycling metabolically active VP2 vesicles carry out the majority of ACh active transport. A method for purifying VP2 vesicles from Torpedo californica electric organ will be developed. The gross physical and chemical properties of VP2 will be compared with those of the well-studied resting VP1 vesicles, and mechanistic aspects of the ACh storage process will be reinvestigated in VP2 vesicles. The VP2 vesicle polypeptides containing the ACh and drug inhibition binding sites of the ACh transport system will be identified by radioactive affinity labeling experiments. The proton pump ATPase which drives ACh storage, drug receptor and ACh transporter proteins will be purified in native detergent solubilized form. The isolated proteins will be reconstituted into liposomes and the ionic mechanism for each wll be characterized. Biochemical properties of each protein will be compared for the forms isolated from VP1 and VP2 vesicles in an effort to identify and localize regulation in the ACh storage system. It is expected that the project will develop the necessary background for biochemical study of ACh storage in mammalian brain and other organs. This should allow development of pharmacologic or other intervention strategies which would effect stimulation of ACh storage in and release from diseased or intoxicated central or peripheral cholinergic nerve terminals. A long-term goal is to find ameliorative strategies of use in the large number of neural dysfunctions, such as Alzheimer's disease, which have been linked to the cholinergic nerve terminal.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS015047-08
Application #
3395916
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1980-03-01
Project End
1993-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Type
Organized Research Units
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Khare, Parul; Ojeda, Ana M; Chandrasekaran, Ananda et al. (2010) Possible important pair of acidic residues in vesicular acetylcholine transporter. Biochemistry 49:3049-59
Efange, Simon M N; Khare, Anil B; von Hohenberg, Krystyna et al. (2010) Synthesis and in vitro biological evaluation of carbonyl group-containing inhibitors of vesicular acetylcholine transporter. J Med Chem 53:2825-35
Luo, Jia; Parsons, Stanley M (2010) Conformational Propensities of Peptides Mimicking Transmembrane Helix 5 and Motif C in Wild-type and Mutant Vesicular Acetylcholine Transporters. ACS Chem Neurosci 1:381-390
Khare, Parul; Mulakaluri, Anuprao; Parsons, Stanley M (2010) Search for the acetylcholine and vesamicol binding sites in vesicular acetylcholine transporter: the region around the lumenal end of the transport channel. J Neurochem 115:984-93
Tu, Zhude; Efange, Simon M N; Xu, Jinbin et al. (2009) Synthesis and in vitro and in vivo evaluation of 18F-labeled positron emission tomography (PET) ligands for imaging the vesicular acetylcholine transporter. J Med Chem 52:1358-69
Khare, Parul; White, Aubrey R; Parsons, Stanley M (2009) Multiple protonation states of vesicular acetylcholine transporter detected by binding of [3H]vesamicol. Biochemistry 48:8965-75
Chandrasekaran, Ananda; Ojeda, Ana M; Kolmakova, Natalia G et al. (2006) Mutational and bioinformatics analysis of proline- and glycine-rich motifs in vesicular acetylcholine transporter. J Neurochem 98:1551-9
Bravo, Dawn T; Kolmakova, Natalia G; Parsons, Stanley M (2005) Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter. Biochemistry 44:7955-66
Bravo, Dawn T; Kolmakova, Natalia G; Parsons, Stanley M (2005) New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates. Neurochem Int 47:243-7
Bravo, Dawn T; Kolmakova, Natalia G; Parsons, Stanley M (2004) Choline is transported by vesicular acetylcholine transporter. J Neurochem 91:766-8

Showing the most recent 10 out of 56 publications