) Abundant evidence has implicated acetylcholine (ACh) in the pathogenesis of Alzheimer's disease (AD). The long term objective of this proposal is to understand the mechanism by which the transport of neurotransmitters (especially ACh) into synaptic vesicles is involved in the processing of neural information and memory and how this may contribute to human disease when it malfunctions. The applicant contributed to the expression-cloning of two vesicular monoamine transporters. Subsequently, genetic studies in the nematode C. elegans identified a putative vesicular ACh transporter (unc-17). This protein shows sequence homology to the vesicular amine transporters and its distribution supports a role in cholinergic transmission. The PI and co-workers have recently isolated homologous transporters from Torpedo californica and rat (rVAChT). Antibody staining of the rat brain tissue analyzed by electron microscopy has shown that rVAChT is indeed a synaptic vesicle-specific integral membrane protein with its C-terminus located in the cytoplasm. Further, the PI has shown that the rVAChT protein binds with high affinity to vesamicol, a drug known to inhibit potently the vesicular acetylcholine transport process, providing further support for the expected function of rVAChT. In addition, ACh competes with vesamicol in a competitive manner, consistent with vesamicol binding to the site of substrate recognition on the rVAChT. The following specific aims are now proposed: I. to identify domains(s) of rVAChT involved in vesamicol and/or ACh binding by generating chimeric vesicular ACh-monoamine transporters using an in vivo chimeric method. The chimeras will be expressed in mammalian cells and membrane fractions from the cells will be analyzed by a reproducible vesamicol binding assay in the presence and absence of ACh. Comparison of binding kinetic values for the chimeras with those of the wild type rVAChT should implicate or identify domain(s)/regions of rVAChT important for vesamicol/ACh binding. II. To generate small deletions and/or point mutations (by site directed mutagenesis) in the rVAChT domains(s) identified to influence vesamicol-/ACh binding. Comparison of binding kinetic values for the mutants with those of the wild type rVAChT should lead to identification of amino acid residues(s) in the rVAChT that are potentially involved in rVAChT and/or ACh binding. Characterizations of many of the point mutations in unc-17 gene, which generate a deficit in neuromuscular function, have led to the conclusion that in C. elegans the unc-17 gene encodes a function essential for survival. Therefore, the proposed studies may proved useful in characterizing the structural basis of rVAChT binding to its inhibitor and substrate and (in the long term) may help explore its mechanism of action and possibly the role of rVAChT and cholinergic transmission in neurodegenerative disorders such as AD.
| Woolf, N J; Milov, A M; Schweitzer, E S et al. (2001) Elevation of nerve growth factor and antisense knockdown of TrkA receptor during contextual memory consolidation. J Neurosci 21:1047-55 |
