Analysis of Point Mutants in the Caenorhabditis
elegans Vesicular Acetylcholine Transporter Reveals Domains
Involved in Substrate Translocation*
Heming
Zhu
,
Janet S.
Duerr§,
Hélène
Varoqui¶,
John R.
McManus§,
James B.
Rand§, and
Jeffrey
D.
Erickson
**
From the Neuroscience Center and Departments of
¶ Ophthalmology and Pharmacology, Louisiana State
University Health Sciences Center, New Orleans, Louisiana 70112 and
§ Program in Molecular and Cell Biology, Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma 73104
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 (Km), 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 vesamicol-insensitive 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.
*
These studies were supported by National Institute of
Neurological Disorders and Stroke Grant NS36936 (to J. D. E.),
National Institute of General Medical Sciences Grant GM38679 (to
J. B. R.), and Oklahoma Center for the Advancement of Science and
Technology Grant HN3-023 (to J. S. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Neuroscience Center,
Louisiana State University Health Sciences Center, 2020 Gravier St.,
Suite D, New Orleans, LA 70112. Tel.: 504-599-0845; E-mail: jerick@lsuhsc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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