The vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) for release from the nerve terminal. A structure-function analysis of VAChT will be done by site-directed mutagenesis, expression and characterization of changes in transporter, inhibitor binding, pH dependence and ACh washout properties of the mutants. A microscopic kinetics model of the VAChT transport cycle will be important to interpretation. The sequences of related vesicular monoamine transporter (VMAT) and bacterial multidrug resistance (mdr) transporters will guide strategy. The goal is to assign specific amino acids to specific functions.
Aim 1 seeks to identify the second of the two proton-translocation sites. The possibility that the core proton-translocation mechanism is not conserved between VAChT and VMAT will be tested in transport-rescue experiments.
Aim 2 seeks to identify the residue of pKa 7.1 that must be deprotonated to bind ACh and the residue of pKa > 9 that must be protonated to transport bound ACh.
Aim 3 seeks to localize the ACh binding site. Also, possible electrostatic guidance for ACh binding will be investigated.
Aim 4 will test whether any of six conserved conformational motifs first identified in bacterial mdr transporters control conformational changes in the VAChT transport cycle. Conserved residues flanking the motifs also will be tested for conformational roles. The project will identify features of VAChT mediating or controlling transport, including those potentially limiting to ACh storage. This might reveal a suitable target for development of a drug that increases ACh storage by modulating VAChT function. The long term goal is effective treatment of cholinergic hypofunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS015047-18
Application #
6393302
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Nichols, Paul L
Project Start
1980-03-01
Project End
2004-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
18
Fiscal Year
2001
Total Cost
$216,730
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

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