The Caenorhabditis elegans choline transporter CHO-1 sustains acetylcholine synthesis and motor function in an activity-dependent manner.
Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.
Matthies
Dawn Signor
DS
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, USA.
Fleming
Paul A
PA
Wilkes
Don M
DM
Blakely
Randy D
RD
D000109:Acetylcholine / Q000096:biosynthesis*; D000222:Adaptation, Physiological; D000818:Animals; D030801:Animals, Genetically Modified; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology / Q000502:physiology; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002794:Choline / Q000493:pharmacokinetics; D018678:Cholinergic Agents / Q000494:pharmacology; D004594:Electrophysiology; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics; D006426:Hemicholinium 3 / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009043:Motor Activity / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013569:Synapses / Q000378:metabolism*; D014018:Tissue Distribution
D029742:Caenorhabditis elegans Proteins; D018678:Cholinergic Agents; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C423186:UNC-104 protein, C elegans; C065637:choline transporter; D049452:Green Fluorescent Proteins; D006426:Hemicholinium 3; D002794:Choline; D000109:Acetylcholine