Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.
Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.
Boulin
Thomas
T
Ecole Normale Supérieure, Biology Department, 75005 Paris, France.
Gielen
Marc
M
Richmond
Janet E
JE
Williams
Daniel C
DC
Paoletti
Pierre
P
Bessereau
Jean-Louis
JL
D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D017173:Caenorhabditis elegans / Q000187:drug effects / Q000235:genetics*; D007978:Levamisole / Q000494:pharmacology*; D011950:Receptors, Cholinergic / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D014982:Xenopus laevis
D000871:Anthelmintics; D011950:Receptors, Cholinergic; D007978:Levamisole