Vesicular acetylcholine transporter (VAChT) moves the neurotransmitter acetylcholine (ACh) from cytoplasm to the inside of synaptic vesicles in cholinergic nerve terminals. This occurs by exchange with protons coming from the inside of the vesicles. The process is inhibited by a drug called vesamicol [(-)- trans-2-(4-phenylpiperidino)cyclohexanol]. Molecular properties of VAChT can be studied by expressing its cDNA in PC12A123.7 cells. Synaptic like microvesicles containing VAChT are prepared form these cells. Vesamicol binding, ACh binding and transport are characterized using radiolabeled ligands and filter assays. A tryptophan in TM VIII has been shown to form part of the ACh binding site and is close to the vesamicol binding site. Also a distant lysine-aspartate ion pair between TMs II and XI, respectively, has been shown to affect ACh and vesamicol affinities by controlling the global conformation of VAChT.
The current study sought to further characterize the ACh and vesamicol binding sites. Residues located close to either the important tryptophan or the ion pair, as determined by a homology model of VAChT, were mutated and functional properties of the mutants were characterized. The results demonstrate that the ACh and vesamicol binding sites indeed are close to the tryptophan. They also demonstrate that the region of the ion-pair is conformationally linked to the ACh and vesamicol binding sites.
The role of a glutamate residue in TM 7 was also investigated. The homology model places it in close contact with a critical proton-carrying aspartate in TM 10. The results of mutations in these two residues demonstrate that the glutamate is not critical to any measured function, as glutamine and aspartate can substitute. Rather, the steric bulk of the glutamate apparently regulates the nearby aspartate, which in turn is critical to a number of VAChT functions.
The vesamicol pH-binding profile shows deviation from the classical bell shape above pH 7. A model to explain the profile has been devised and tested. It requires the presence of three functional weak acids in VAChT. Overall, ACh, vesamicol, and proton binding sites in VAChT have been significantly described at a molecular level.
|Advisor:||Parsons, Stanley M.|
|Commitee:||Gerig, John T., Plaxco, Kevin W., Waite, Herbert|
|School:||University of California, Santa Barbara|
|School Location:||United States -- California|
|Source:||DAI-B 71/02, Dissertation Abstracts International|
|Keywords:||Acetylcholine, Ligand binding, VAChT, Vesicular acetylcholine transporter|
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