Mutations in the Extracellular Domain and in the Membrane-Spanning Domains Interfere with Nicotinic Acetylcholine Receptor Maturation

Abstract

The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking.

Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans

“…Reconstruction of the C. elegans nervous system from electron micrographs (EM) of serial sections suggested a relatively simple PVD architecture comprised of elongated, unbranched lateral processes projecting from anterior and posterior sides of each PVD soma and a single axon that grows downward to enter the ventral nerve cord (White et al, 1986). However, images of PVD obtained in the light microscope after immunostaining for a PVD-expressed membrane receptor (Halevi et al, 2002; Yassin et al, 2001; Yassin et al, 2002) or with a PVD-specific GFP reporter revealed a much more elaborate morphology with many additional dendritic branches(Tsalik and Hobert, 2003). Here we have used a bright PVD::GFP marker ( F49H12.4:: GFP) (Watson et al, 2008) (Fig 1) to reveal that PVD architecture is defined by a complex but well-ordered array of non-overlapping sister dendrites and that the creation of this structure involves a stereotypical series of branching decisions.…”

RIC‐3: a nicotinic acetylcholine receptor chaperone

“…It was assumed that screening for mutations that cause suppression of the deg‐3(u662) phenotype might identify genes, which are required for nAChR function. Several suppressor mutations were identified within the deg‐3 gene itself ( Halevi et al , 2002 ; Yassin et al , 2002 ). Other suppressor mutations were identified within des‐2 ( Halevi et al , 2002 ), which encodes a nAChR subunit (DES‐2) that co‐assembles with DEG‐3 ( Treinin et al , 1998 ; Yassin et al , 2001 ).…”

Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans

“…Analysis of deg-3 mutants in combination with homology modelling of the DEG-3 receptor have suggested that several amino acid residues in the Nterminal region are important for correct receptor maturation, assembly and localization by, for example, maintaining correct protein folding. (84) The study of ligand-receptor interactions using site-directed and in silico mutagenesis has proven successful in implicating amino acid residues important in determining imidacloprid specificity to insect nAChRs over vertebrate receptors. (85) Thus, further studies of this kind, in combination with pharmacological analysis of nAChRs expressed in heterologous systems such as Xenopus oocytes, should greatly assist in the development of novel drugs/ chemicals for use in human health, animal health and agriculture.…”