Vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) in synaptic vesicles for evoked ?elease from cholinergic nerve terminals. It is allosterically inhibited by a compound called vesamicol. The project seeks understanding of VAChT structure and transport mechanism with the long term goal of developing a pharmacology to control ACh storage (and thus release) for treatment of cholinergic disorders. Experiments using site-directed mutagenesis, expression and characterization of mutant properties are proposed. A residue in VAChT must be protonated for transport.
The first aim i s to test whether the recent Dreliminary assignment of the aspartate residue in putative transmembrane domain XI (TMD XI) to this role s correct. A different aspartate or glutamate residue that recently was shown not to be in a TMD must be deprotonated to bind ACh and vesamicol.
The second aim i s to identify it.
The third aim i s to test and refine the recent assignments of ACh and vesamicol binding sites to putative TMDs XIII and X. The aspartate residue in putative TMD X is known to participate in proton translocation reactions that provide energy input to VAChT.
The fourth aim i s to test and refine the proposed role(s) of this residue using a novel assay that monitors transmembrane reorientation of the ACh binding site. The fifth aim is to test whether VAChT contains 12 TMDs, as is now assumed, by inserting one cysteine residue into each hydrophilic region of the sequence and determining which side of the membrane the residue is exposed to using a chemical labeling technique. The sixth aim is to identify which hydrophilic regions change conformation when VAChT binds ACh and vesamicol and transports ACh by using the labeling technique under conditions that detect different rates of labeling. The experiments will be extended to mutants of residues responsible for the required protonation and deprotonation to identify which hydrophilic regions change conformation at pH extremes. The results will provide substantial new information about VAChT structure and dynamics.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Silberberg, Shai D
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University of California Santa Barbara
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Santa Barbara
United States
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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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