The packaging of biogenic amines and acetylcholine into specialized secretory vesicles of neurons and neuroendocrine cells is mediated by specific vesicular transporters. Two isoforms of the human vesicular monoamine transporter (hVMAT1 and hVMAT2) and the vesicular Ach transporter (hVAChT) have recently been cloned and functional assays for each have been developed. Because these transporters are highly related in both structure and function, the analysis of their substrate and inhibitor binding sites should provide insight into the mechanisms of vesicular transport and into the means by which transmitter release is affected by altered transporter properties. The major goals of this proposal are to define the molecular structures that determine transporter specificity and to test the hypothesis that vesicular transporters may be a potential site for regulation of synaptic function. In order to define the transmembrane domains (TMD) of the vesicular transporter proteins which participate in the substrate translocation pore and the molecular structures that determine transporter specificity, chimeric transporter and site-specific mutants will be used to test the following hypotheses: 1) two substrate binding sites exist on these transporters: they include a high-affinity cytoplasmic recognition site and a low-affinity discharge site located towards the vesicular lumen. 2) distinct amino acid residues are involved in the differential recognition properties of hVMAT1 and hVMAT2 to high-affinity and low-affinity unsubstituted aromatic amines. 3) VMAT1 and VMAT2 can functionally interact, perhaps as a dimer, and express altered transporter specificity, 4) the substrate binding sites of hVMAT1 and hVAChT reside in different TMDs. To test the hypothesis that alterations in the molecular structures that determine vesicular transporter specificity can affect the levels of neurotransmitter available for regulated neurosecretion, the following predictions are made: 1) site-specific mutations in hVAChT that are based on several non-lethal unc-17 point mutations in C. elegans will affect the kinetic parameters of uptake and steady-state level of vesicular ACh accumulation in vitro, and 2) changes in the affinity of ACh for VAChT or the level of expression of VAChT will affect the amount of ACh accumulated in synaptic vesicles and the amount of ACh released from cholinergic neurons in culture.
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