Analysis of point mutants in the Caenorhabditis elegans vesicular acetylcholine transporter reveals domains involved in substrate translocation

Cholinergic neurotransmission depends upon the regulated release of acetylc holine. This requires the loading of acetylcholine into synaptic vesicles b y the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of t he VAChT gene of Caenorhabditis elegans (CeVAChT) (unc-17) and exhibit beha vioral phenotypes consistent with a reduction in vesicular transport activi ty and neurosecretion. Several of these mutants express normal amounts of V AChT protein and exhibit appropriate targeting of VAChT to synaptic vesicle s. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stabl y expressed these cDNAs in PC-12 cells. These mutants display selective def ects in initial acetylcholine ;transport velocity (K-m), with values rangin g from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The rela tive order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specifi c alterations in the intrinsic parameters of transport and in the storage a nd synaptic release of acetylcholine in vivo.