Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor

Abstract

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligandgated 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 ␣-and 2 non-␣-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.anthelminthic drug ͉ recombinant receptor expression

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled α-subunits

“…Newly synthesized subunits interact with the general folding machinery including calnexin and BIP (16). In Caenorhabditis elegans, three genes are essential for functional reconstitution of the levamisole-sensitive AChR (L-AChR) (17). One of them, UNC-50, is important for forward trafficking (18).…”

The orphan pentameric ligand-gated ion channelpHCl-2is gated by pH and regulates fluid secretion inDrosophilaMalpighian tubules

“…The subunits of pLGICs have a stereotypical tertiary structure that consists of three general domains: an aminoterminal extracellular ligand-binding domain, four transmembrane domains (M1-M4), which collectively form the ion-permeable channel pore, and an intracellular loop between M3 and M4 (Dent, 2006;Hille, 2001;Sine and Engel, 2006;Tasneem et al, 2005). Functional channels can exist as homomers, or as heteromers, containing as many as five distinct channel subunits (Boulin et al, 2008).…”

Contribution of Subunits toCaenorhabditis elegansLevamisole-Sensitive Nicotinic Receptor Function

“…Boulin et al (2008) reconstituted functional L-AChRs in Xenopus laevis oocytes by coexpressing the five different LAChR subunits together with three ancillary proteins. ACR-8, an ␣-type subunit highly homologous to LEV-8 (Mongan et al, 1998), is also present in muscle but its function remains unclear (Gottschalk et al, 2005;Touroutine et al, 2005).…”