A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans

Abstract

Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates.

Figure 1

Phenotypic characterization of lev-10 mutants. a, The levamisole dose–response curve indicates that lev-10 mutants are only weakly resistant to levamisole when compared with unc-29(x29) mutants, which lack levamisole-sensitive AChRs. Error bars represent s.e.m. (n = 4 independent experiments). WT, wild type. b, lev-10 mutants exhibit weak locomotory defects compared with the wild type in a thrashing assay (ANOVA test; p < 0.01) but are not as impaired as unc-29(x29) mutants (p < 0.01). Error bars represent s.e.m. (n = 6).

Figure 2

Mutation of lev-10 results in the specific loss of levamisole-sensitive AChR clusters at neuromuscular junctions. a–d, UNC-29 localization detected by immunofluorescence with anti-UNC-29 antibodies. a, Shown are the nerve ring (nr) and the dorsal (dc) and ventral (vc) nerve cords in wild-type animals. c, Individual UNC-29 puncta at high magnification in the dorsal cord from the wild type. b, d, UNC-29 staining in lev-10(kr26) animals at magnifications as in a and c, respectively. The staining in the pharynx is non-specific (data not shown). e, Visualization of cholinergic varicosities by co-immunostaining of the vesicular ACh transporter UNC-17 in wild-type animals shows that UNC-29 clusters are juxtaposed to cholinergic release sites (arrowheads). f, UNC-17 staining in lev-10(kr26) mutants. g, h, Immunostaining of the GABA receptor UNC-49 in wild-type animals (g) and lev-10(kr26) mutants (h). Scale bars, 20 μm. i, Western blot with anti-UNC-29 and anti-VHA-5 antibodies on membrane fractions of C. elegans extracts. The UNC-29 protein has an apparent molecular mass of 47 kDa. VHA-5 detection is used for normalization. WT, wild type.

Figure 3

Levamisole-sensitive AChRs are functional but diffusely distributed in lev-10 body-wall muscle. a, Currents recorded from voltage-clamped body wall muscles in response to pressure-ejection of levamisole (300 μM) in wild-type (WT) and lev-10(kr26) mutants. b, Average amplitude of levamisole-elicited current. c, Evoked currents recorded in a body-wall muscle after eliciting neurotransmitter release by ventral nerve cord depolarization. Experiments were performed in an unc-49(e407) background to eliminate currents due to GABA receptor activation and in the presence of 5 μM DHβE, which blocks the levamisole-insensitive AChRs. d, Average amplitude of evoked response. Error bars in b and d represent s.e.m.

Figure 4

lev-10 encodes a CUB domain-rich transmembrane protein. a, Genomic organization of lev-10. Open boxes, coding regions; black boxes, 5′ and 3′ untranslated region; ATG, translational start site; SL1, SL1 trans-spliced leader. The first intron of lev-10 contains the first exon of the gene eat-18 (hatched boxes). The eat-18 exon is spliced to the second exon of lev-10 by using a different frame, which ends 16 bp after the splice site. ad1110, nonsense mutation in the first exon of eat-18. b, Predicted structure of the LEV-10 isoforms. Horizontal black line, signal peptide; CUB, complement, urchin epidermal growth factor, and bone morphogenetic protein domain; LDLa, low-density lipoprotein receptor domain class A; TM, transmembrane region; aa, amino acids. Domain predictions were based on SMART (smart.embl-heidelberg.de).

Figure 5

LEV-10 is a synaptic protein that requires levamisole-sensitive AChRs for proper localization but not for expression. a, LEV-10A immunostaining in the dorsal cord of a wild-type animal. c, UNC-17 immunostaining of the same animal labels cholinergic varicosities. e, Merged images. f, Z-optical projection through the entire stack of confocal images at the level of the dashed arrow in e. b, d, LEV-10A (b) and UNC-17 (d) immunostaining in the dorsal cord of an unc-29(x29) mutant. Scale bar, 10 μm. g, Western blot analysis of fractionated C. elegans extracts. P, pellet; S, cytosolic supernatant. The LEV-10 transmembrane protein has an apparent molecular mass of about 120 kDa. Mutants of the levamisole-sensitive AChR subunits unc-29(x29) and unc-38(x20) have LEV-10 concentrations at the membrane comparable to those of the wild type.