Analysis of Point Mutants in the Caenorhabditis elegans Vesicular Acetylcholine Transporter Reveals Domains Involved in Substrate Translocation

Abstract

Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans (CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (K m ), with values ranging 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 vesamicolinsensitive uptake of acetylcholine. The relative 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 specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

“…The large increase in the ACh content is opposite from what happens in mice null for vesicular monoamine transporter 2, as they have a decrease in intracellular monoamine contents (62). C. elegans carrying a blocking mutation of VAChT also has an increase in ACh contents (29,65).…”

“…VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD HOM ] mice) are myasthenic and have cognitive deficits (47).…”

Excitation-Transcription Coupling via Calcium/Calmodulin-dependent Protein Kinase/ERK1/2 Signaling Mediates the Coordinate Induction of VGLUT2 and Narp Triggered by a Prolonged Increase in Glutamatergic Synaptic Activity

“…After 18 h, cells were treated with sodium butyrate (5 mM) to increase transgene expression (51). Cells were harvested Ͻ48 h post-transfection.…”

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

“…Most expression assays for VGLUTs are performed with light vesicle preparations from stably-transformed neuroendocrine PC12 or BON cell lines. Although we have developed a transient assay in PC12 cells to assess VGLUT function that is less cumbersome than that involving generation of stable cell lines when analyzing various functional mutants [26,53], we sought here to develop a convenient, costeffective, and improved vesicular glutamate transport assay using Xenopus oocytes. Xenopus oocytes have primarily been an excellent tool for the expression cloning and functional characterization of transporters or receptors that function on the plasma membrane [40,54].…”