An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse
2009; Springer Nature; Volume: 28; Issue: 17 Linguagem: Inglês
10.1038/emboj.2009.204
ISSN1460-2075
AutoresRuta B Almedom, Jana Liewald, Guillermina Hernando, Christian Schultheis, Diego Rayes, Jie Pan, Thorsten Schedletzky, Harald Hutter, Cecilia Bouzat, Alexander Gottschalk,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle16 July 2009free access An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse Ruta B Almedom Ruta B Almedom Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Jana F Liewald Jana F Liewald Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Guillermina Hernando Guillermina Hernando Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Christian Schultheis Christian Schultheis Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Diego Rayes Diego Rayes Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Jie Pan Jie Pan Department of Biological Sciences, Simon Fraser University, University Drive, Burnaby, British Columbia, Canada Search for more papers by this author Thorsten Schedletzky Thorsten Schedletzky Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Harald Hutter Harald Hutter Department of Biological Sciences, Simon Fraser University, University Drive, Burnaby, British Columbia, Canada Search for more papers by this author Cecilia Bouzat Cecilia Bouzat Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Alexander Gottschalk Corresponding Author Alexander Gottschalk Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Cluster of Excellence Frankfurt—Macromolecular Complexes (CEF-MC), Goethe-University, Frankfurt, Germany Search for more papers by this author Ruta B Almedom Ruta B Almedom Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Jana F Liewald Jana F Liewald Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Guillermina Hernando Guillermina Hernando Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Christian Schultheis Christian Schultheis Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Diego Rayes Diego Rayes Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Jie Pan Jie Pan Department of Biological Sciences, Simon Fraser University, University Drive, Burnaby, British Columbia, Canada Search for more papers by this author Thorsten Schedletzky Thorsten Schedletzky Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Search for more papers by this author Harald Hutter Harald Hutter Department of Biological Sciences, Simon Fraser University, University Drive, Burnaby, British Columbia, Canada Search for more papers by this author Cecilia Bouzat Cecilia Bouzat Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina Search for more papers by this author Alexander Gottschalk Corresponding Author Alexander Gottschalk Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany Cluster of Excellence Frankfurt—Macromolecular Complexes (CEF-MC), Goethe-University, Frankfurt, Germany Search for more papers by this author Author Information Ruta B Almedom1,‡, Jana F Liewald1,‡, Guillermina Hernando2, Christian Schultheis1, Diego Rayes2, Jie Pan3, Thorsten Schedletzky1, Harald Hutter3, Cecilia Bouzat2 and Alexander Gottschalk 1,4 1Department of Biochemistry, Chemistry and Pharmacy, Johann Wolfgang Goethe-University, Institute of Biochemistry, Frankfurt, Germany 2Instituto de Investigaciones Bioquimicas, Universidad Nacional del Sur-CONICET, Bahia Blanca, Argentina 3Department of Biological Sciences, Simon Fraser University, University Drive, Burnaby, British Columbia, Canada 4Cluster of Excellence Frankfurt—Macromolecular Complexes (CEF-MC), Goethe-University, Frankfurt, Germany ‡These authors contributed equally to this work *Corresponding author. Department of Biochemistry, Johann Wolfgang Goethe-University Frankfurt, Cluster of Excellence Frankfurt—Macromolecular Complexes, Max-von-Laue-Str. 9, Frankfurt 60438, Germany. Tel.: +49 69 7982 9261; Fax: +49 69 7982 9495; E-mail: [email protected] The EMBO Journal (2009)28:2636-2649https://doi.org/10.1038/emboj.2009.204 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive’ nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. Introduction Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentamers composed of α- and non-α-subunits, which mediate fast synaptic transmission in neurons and muscles (Changeux and Edelstein, 2005). The agonist binds at the interface between an α-subunit and either another α- or a non-α-subunit (Chiara and Cohen, 1997). Two or three acetylcholine (ACh) molecules need to bind for maximal activation (Karlin, 2002; Rayes et al, 2009); thus, functional properties of nAChRs are affected by the number of α-subunits, and the presence of particular subunits in the pentamer. In vertebrates, α-, β-, δ-, γ- and ε-subunits are found in muscle, and nAChRs are of α2βδγ or α2βδε composition, depending on the developmental stage (Mishina et al, 1986); in neurons, 9 α- and 3 β-subunits form α5- or α2β3-type receptors. The nAChR subunit repertoire of Caenorhabditis elegans is even more complex: its genome encodes 29 confirmed nAChR subunits (Jones et al, 2007), of which at least seven are expressed in muscle, based on microarray profiling and biochemical purification (Gottschalk et al, 2005; Touroutine et al, 2005; Fox et al, 2007). However, expression of additional nAChRs in muscle was demonstrated (Treinin et al, 1998). Regulating nAChR subunit composition is an important way to fine-tune cholinergic signalling. Subunit combinations can be predetermined by cell-specific expression, and many potential assembly intermediates may be unstable due to incompatible subunit interfaces. In vertebrate neurons, a vast variety of nAChRs could be generated; however, only few combinations were detected experimentally (Gotti et al, 2007). Out of the 208 possible combinations of vertebrate muscle nAChR subunits, only one is found in mature muscle. To some extent, this is explained by sequence-specific interactions within the N-terminal, as well as the first transmembrane (TM) domains, according to different models (Gu et al, 1991; Kreienkamp et al, 1995; Wang et al, 1996; Keller and Taylor, 1999; Wanamaker et al, 2003). HSP70 chaperones and the ER quality control assist in nAChR assembly (Blount and Merlie, 1991; Keller et al, 1996, 1998; Keller and Taylor, 1999). The ER-resident TM protein RIC-3 and the Golgi-associated protein UNC-50 are also required for efficient nAChR assembly, maturation or trafficking from the ER and beyond (Halevi et al, 2002; Eimer et al, 2007), and 14-3-3 proteins further assist nAChRs in leaving the ER (Jeanclos et al, 2001). Immature assemblies and single subunits are retained in the ER, as they expose retention motifs in the first TM helix, which are masked only on closed pentamer formation (Wang et al, 2002). Yet, no factors are known that select particular subunits for incorporation into mature receptors, particularly in cells expressing many different nAChR subunits. It is further unknown whether there is active sorting that allows only particular nAChRs to exit the ER. The C. elegans ‘levamisole-sensitive’ nAChR (L-AChR) is expressed in muscle cells, but some of its subunits are also found in neurons. Genetic screens based on levamisole-induced paralysis defined three essential subunits: UNC-38, UNC-63 (both α-subunits) and UNC-29 (non-α; Lewis et al, 1987; Fleming et al, 1997; Culetto et al, 2004). Additional L-AChR subunits, LEV-8 (α) and LEV-1 (non-α), are considered non-essential as their loss confers weak levamisole resistance (Lewis et al, 1987; Culetto et al, 2004; Towers et al, 2005). Co-expression of these five subunits in Xenopus oocytes, together with essential L-AChR biogenesis factors, RIC-3, UNC-50 and UNC-74, sufficed to constitute levamisole-activated currents (Boulin et al, 2008). An electrophysiologically defined ‘nicotine-sensitive’ N-AChR contributes to ACh currents at neuromuscular junctions (NMJs). This apparently homopentameric receptor consists of ACR-16 subunits (Francis et al, 2005; Touroutine et al, 2005). To define proteins contributing to L-AChR function, we previously purified the L-AChR by tandem affinity purification and identified co-purified proteins by mass spectrometry (Gottschalk et al, 2005). In addition to the five genetically identified L-AChR subunits, we found two more α-subunits, ACR-8 and ACR-12. Although ACR-12 is expressed in neurons only, ACR-8 is expressed in body wall muscle cells. Thus, seven nAChR subunits are implicated in L-AChR function in vivo, suggesting that L-AChRs may represent a mixed population of pentamers with variable subunit composition, and/or that their composition could depend on the particular cell. Non-nAChR proteins that co-purified with the L-AChR were screened for effects on the in vivo sensitivity to cholinergic agonists (Gottschalk et al, 2005). Among proteins causing reduced agonist sensitivity was the product of gene T05F1.1, subsequently termed nra-2. Here, we show that NRA-2, in complex with a second protein, NRA-4, acts in the ER to affect functional properties and subunit composition of L-AChRs expressed at synapses. Electrophysiological properties of L- and N-AChRs are altered in nra-2 and nra-4 mutants, as well as single-channel L-AChR properties in embryonic muscle, consistent with faster desensitization of L-AChRs. Synaptic expression of UNC-29 and, particularly, UNC-38 subunits are characteristically altered in nra-2 and nra-4 mutants. Mutations in acr-8 suppress nra-2 phenotypes, and synaptic expression of ACR-8 is increased in nra-2 mutants, uncovering a reciprocal regulation of UNC-38 versus ACR-8 α-subunit incorporation into synaptic nAChRs by NRA-2. Thus, NRA-2 and NRA-4 affect L-AChR properties by altering subunit composition and/or the relative abundance of particular L-AChR subtypes at the synapse. Results NRA-2 and NRA-4 are type I TM proteins associated with L-AChRs NRA-2 is a type I TM protein, consisting of a 518 amino acid (aa) luminal domain and an 18 aa cytosolic tail (Figure 1A and B), and contains a peptidase domain, likely inactive, as certain amino acids are non-conserved (Supplementary Figure 1). NRA-2 resembles vertebrate Nicalin (nicastrin-like protein; Supplementary Figures 2 and 3). Nicastrins are subunits of the integral membrane peptidase γ-secretase (Yu et al, 2000). Nicalin, which is not part of γ-secretase, antagonizes TGFβ signalling in an ill-defined manner, acting in complex with a second type I TM protein, termed NOMO (nodal modulator) in the ER (Haffner et al, 2004, 2007). Nicalin and NOMO were shown to stabilize each other in this complex. Interestingly, the C. elegans homologue of NOMO (gene C02E11.1; Figure 1A; Supplementary Figures 4 and 5), was among the proteins we co-purified with the L-AChR (Gottschalk et al, 2005). We termed this protein NRA-4. NRA-4 has a 1068 aa luminal domain, a 30 aa cytosolic tail and no motifs suggesting a function (Figure 1B). Both nra-2 and nra-4 produce only single-splice variants, based on published ESTs (www.wormbase.org) and sequencing of full-length cDNAs obtained from Y Kohara. Deletion alleles of nra-2 (tm1453 and ok1731) and nra-4 (hd127 and tm2656) were obtained for further study (Figure 1; Supplementary Figures 2 and 4). Figure 1.Cholinergic agonist-induced phenotypes are altered in nra-2 and nra-4 mutants, and rescued by muscle-specific expression. (A) The nra-2 and nra-4 genes, as annotated in www.wormbase.org, were confirmed by sequencing cDNAs kindly provided by Y Kohara. Sequences deleted in the alleles used are indicated by bars. (B) The nra-2 and nra-4 genes encode predicted type I TM proteins with signal sequences (SS), thus they are expected to be synthesized into the ER lumen, exposing a short C-terminal cytosolic tail. Deletion/insertion alleles tm1453 and ok1731 truncate NRA-2, bringing stop codons (X) in frame. nra-4(hd127) removes part of the promoter and exon I including SS and start codon and tm2656 is a predicted in-frame deletion. (C, D) Paralysis time-course of wild-type and mutant animals exposed to 0.2 or 0.25 mM levamisole (C) or 31 mM nicotine (D). The fraction of non-paralyzed animals was counted every 15 min. Experiments were repeated 3–7 times (30 animals tested each time), data represent mean±s.e.m., statistically significant differences to wild type are indicated (*P<0.05; **P<0.01; ***P<0.001). Brackets indicate overall significant differences between genotypes, if they were different for at least three time points. (E) Swimming cycles of animals immersed for 1 h in M9 buffer with 8 mM muscimol, a GABAAR agonist, were normalized to swimming cycles of untreated control animals. Download figure Download PowerPoint Mutants in nra-2(ok1731) were slightly uncoordinated, and nra-2(ok1731) and nra-4(tm2656) mutants showed reduced brood size (data not shown). The nra-2 alleles truncate the NRA-2 protein C-terminal, leaving only 294 (tm1453) or 212 (ok1731) aa of the luminal domain (Supplementary Figure 6). Alleles of nra-4 delete N- (hd127) or C-terminal (tm2656) sequences. hd127 is predicted to remove 183 nt of the promoter and the first 48 aa, including a leader sequence (Figure 1; Supplementary Figure 6). As the second exon, unaffected by hd127, begins with an ATG, a protein without leader sequence could be made. RT–PCR analysis confirmed the presence of an nra-4 transcript lacking exon 1 in hd127 mutants (data not shown). However, it is unclear whether the truncated promoter expresses in the same tissues as the full-length promoter, or whether any functional protein is made in this mutant. The nra-4(tm2656) allele removes aa 816–920 of the luminal domain in-frame, leaving TM domain and cytosolic tail intact (Figure 1; Supplementary Figure 6). As most assays used in this work showed no phenotypes of nra-4(tm2656), we consider it at most a reduction-of-function allele (see Supplementary Figure 7 for a summary of experiments involving nra-4(tm2656)). NRA-2 and NRA-4 affect in vivo sensitivity to cholinergic, but not GABAergic agonists, and act cell autonomously in muscle We tested the nra-2 and nra-4 mutants in paralysis assays for altered in vivo sensitivity to cholinergic agonists (nicotine and levamisole), and to aldicarb, an ACh-esterase inhibitor that causes ACh accumulation in the synaptic cleft. Both alleles of nra-2 as well as nra-4(hd127) caused mild resistance to either drug, indicating reduced activity of muscle nAChRs (Figure 1C and D; Supplementary Figure 8). The paralysis phenotypes could be reversed by expression of the nra-2 cDNA in muscle only (using pmyo-3), and nra-4 under its own promoter, in the respective mutants (Figure 1C and D; Supplementary Figure 9A). Thus, at least NRA-2 acts cell autonomously in muscle. Double mutants of nra-2 and nra-4 (and double RNAi; data not shown) showed no exacerbation of the single-mutant effects in paralysis assays, indicating that NRA-2 and NRA-4 act in the same pathway. To test whether NRA-2 and NRA-4 generally affect ligand-gated ion channels at the NMJ, we assayed function of the inhibitory GABAA receptor. Swimming behaviour was analysed in the presence of muscimol, a GABAAR agonist that slows down swimming rate. Muscimol sensitivity was unaffected in nra-2, nra-4 or nra-2; nra-4 double mutants, indicating that nra-2 and nra-4 do not act on GABAARs (Figure 1E). Human Nicalin partially functions in C. elegans, likely independent of TGFβ signalling The Nicalin/NOMO ER protein complex was shown to act in signalling through the nodal TGFβ pathway, but a potential function in vertebrate nAChR biology was not investigated (Haffner et al, 2004). We thus asked whether human Nicalin could rescue nra-2 cholinergic phenotypes. Human Nicalin cDNA, fused to GFP, was expressed in muscle cells of nra-2(ok1731) mutants, which caused partial rescue of levamisole and nicotine resistance phenotypes (Supplementary Figure 9B), indicating potential conservation of an nAChR-associated function of Nicalin. However, transgenic animals were small, slightly uncoordinated, and Nicalin∷GFP partially aggregated (Supplementary Figure 9C), possibly preventing full rescue. As nra-2 and nra-4 mutants may affect cholinergic signalling indirectly through TGFβ pathways, we tested mutants in these pathways for cholinergic phenotypes. C. elegans has five TGFβ ligands (Savage-Dunn, 2005): two are of unknown function, DAF-7 controls the dauer larval state (Ren et al, 1996), whereas DBL-1 affects body size (Suzuki et al, 1999) and GABA signalling at the NMJ (Vashlishan et al, 2008), neither of which is altered in nra-2 or nra-4 mutants. UNC-129 affects dorsoventral axon guidance of some motor neurons, and could thus affect the NMJ (Colavita et al, 1998). We analysed levamisole and nicotine paralysis in the mutants unc-129(ev554), dbl-1(wk70), daf-7(e1372) and in TGFβ receptor mutants daf-1(m402) and sma-6(wk7) (Supplementary Figure 10A and B). dbl-1(wk70) and sma-6(wk7) animals were hypersensitive to nicotine and levamisole. For dbl-1, this was previously shown to be caused by a GABA signalling defect (Vashlishan et al, 2008). sma-6(wk7) mutants were sick and paralyzed immediately, likely indicating a cuticle defect. The other mutants had normal sensitivity to cholinergic agonists. Effects of nra-2 and nra-4 alleles on TGFβ signalling are most likely not causing the observed cholinergic defects, though we cannot completely rule out that the two TGFβ ligands of unknown function may affect NMJs. NRA-2 and NRA-4 form a protein complex in the ER and co-localize with the L-AChR NRA-2 and NRA-4 may affect nAChR biogenesis and/or function either in the ER, in which the vertebrate homologues form a complex, in the Golgi, the secretory pathway or at synapses. To determine the site of action of these proteins, we analysed their subcellular localization using fluorescent proteins as tags. NRA-2∷GFP, NRA-2∷mCherry and NRA-4∷GFP showed a reticular pattern reminiscent of the ER in muscles (for NRA-2 and NRA-4; Figures 2 and 3) and other cells (for NRA-4∷GFP only; Figure 2B; pnra-4 and pnra-2 are active in muscles, neurons and other tissues; Supplementary Figure 11). NRA-2∷GFP also co-localized with an ER marker in HeLa cells (data not shown). To study whether NRA-2 and NRA-4 physically interact in vivo, we used bimolecular fluorescence complementation (BiFC; Chen et al, 2007; Shyu et al, 2008). Indeed, NRA-2 and NRA-4 interact within the ER membrane (Figure 2C), whereas NRA-4 and an unrelated control membrane protein, the stomatin UNC-1, do not (Figure 2D). Thus, NRA-2 and NRA-4 form a membrane protein complex in the ER of muscle cells, in which they may interact with nAChRs during biogenesis and assembly. Figure 2.NRA-2 and NRA-4 are expressed in the ER and interact in a complex. (A) NRA-2∷YFP (upper panel, single confocal plane) or NRA-2∷GFP (lower panel, epifluorescence) were expressed from the muscle-specific pmyo-3 promoter. Reticular expression, reminiscent of the ER was found. (B) NRA-4∷GFP was expressed from the endogenous pnra-4 promoter. Intracellular, reticular expression was observed in muscle cells (upper panel) and neurons (arrowhead), and in other tissues (lower panel: muscles, neurons and hypodermal cells in the tail). (C) NRA-2 and NRA-4 form a complex, as shown by bimolecular fluorescence complementation (BiFC). NRA-2 was fused to the VN173 fragment of Venus, and NRA-4 to the VC155 fragment. Fluorescence was restored in muscle ER (arrows point to muscle cell nuclei surrounded by ER), in which the two proteins were co-expressed. (D) NRA-4∷VC155 does not interact in the ER with the stomatin UNC-1∷VN173, expressed in muscle (a gift by ZW Wang). Occasionally, vesicular fluorescent structures were observed, possibly representing lysosomes in which the fusion proteins are degraded and in whose membranes their cytosolic tails (and Venus fragments) accumulate. Size bars are 10 μm. Download figure Download PowerPoint Figure 3.NRA-2 co-localizes with L-AChR subunits in the ER, but not at synapses. (A) NRA-2∷mCherry (expressed from the pmyo-3 promoter) was co-expressed with the L-AChR subunit UNC-29∷GFP (expressed from punc-29) and co-localization was observed by confocal microscopy (single confocal plane of midbody muscle cells). (B) Endogenous UNC-29 protein was immunolabelled with specific antibodies in animals expressing NRA-2∷GFP in muscles (GFP fluorescence was preserved during fixation). Dorsal nerve cord (dnc) and adjacent muscle cells (bwm) are shown near the pharyngeal terminal bulb. No co-localization of NRA-2∷GFP and UNC-29 was apparent. (C) NRA-2∷GFP was co-expressed in muscle with epitope-tagged UNC-38∷3xMYC (expressed from punc-38). UNC-38, exposing the MYC tag on the cell surface, was labelled with Cy3-conjugated anti-MYC antibodies injected into the body cavity. The ventral nerve cord was imaged by confocal microscopy (single focal plane), showing punctate cell-surface L-AChR clusters that contain UNC-38. NRA-2∷GFP is adjacent to L-AChR clusters, but not co-localizing with them (inset: enlarged region). (D) SEC-23∷GFP, a COPII coat component that labels ER exit sites, and NRA-2∷mCherry were co-expressed in muscle and imaged by confocal microscopy. Puncta of SEC-23 accumulation contained also NRA-2; however, NRA-2 did not accumulate at these sites. Z-stack of confocal sections. Size bars are 10 μm. Download figure Download PowerPoint Consistent with this idea, NRA-2∷mCherry and the L-AChR subunit UNC-29∷GFP largely co-localized in ER membranes (Figure 3A). Although L-AChR subunits are visible in the ER only when over-expressed (endogenous L-AChRs are only detectable at synapses; Figure 3B; Gally et al, 2004), a diffuse localization of nascent nAChRs in the ER is not unexpected. Several additional observations argue against direct interactions of NRA-2/NRA-4 with L-AChRs at synapses: (1) NRA-2∷GFP and NRA-4∷GFP did not accumulate at the plasma membrane or the tips of muscle arms, in which NMJ postsynaptic elements are found (Gottschalk et al, 2005; Gottschalk and Schafer, 2006; Eimer et al, 2007). (2) The endogenous L-AChR subunit UNC-29 does not co-localize with NRA-2∷GFP (Figure 3B). (3) NRA-2∷GFP does not co-localize with the synaptic UNC-38∷3xMYC L-AChR subunit (Figure 3C; the latter one immunolabelled at the cell surface, using fluorescent antibodies injected into the body cavity; Gottschalk et al, 2005; Gottschalk and Schafer, 2006; Eimer et al, 2007). (4) Minor amounts of cell-surface NRA-2 were detected with extracellular anti-HA antibody in animals expressing 3xHA∷NRA-2∷GFP, in clusters along muscle cell boundaries (Supplementary Figure 12), but this did not accumulate at nerve cords, in which synaptic L-AChRs are found. Cell-surface expression of 3xHA∷NRA-2∷GFP may be due to overexpression (its binding partner NRA-4 was not overexpressed). In sum, our observations do not support an interaction of NRA-2 with L-AChRs at synapses. NRA-2 may interact with L-AChRs during assembly, or when they are sorted for ER exit. However, NRA-2∷mCherry and SEC-23∷GFP, a COPII coat component localizing to ER exit sites and secretory vesicles (Roberts et al, 2003) showed different localization patterns: SEC-23∷GFP was found in punctate intracellular clusters, whereas NRA-2∷mCherry was not enriched at these sites (Figure 3D). Thus, NRA-2 is likely not part of the ER exit machinery. Cholinergic inward currents in muscle cells are reduced in nra-2 and nra-4 mutants To directly measure nAChR and GABAAR function in muscle, we recorded postsynaptic currents (PSCs) evoked by pressure-applied ACh, levamisole, nicotine and GABA under whole-cell voltage-clamp (Supplementary Table 1; Richmond and Jorgensen, 1999; Francis et al, 2003; Richmond, 2006; Liewald et al, 2008). Levamisole- and nicotine-evoked PSCs were significantly reduced in both nra-2 mutants (ok1731: levamisole: 62±6%, normalized to wild type, P<0.01, t-test; nicotine: 57±5%, P<0.001; tm1453: levamisole: 74±9%, P<0.05; nicotine: 72±8%, P<0.05), as well as in nra-4(hd127) mutants (levamisole: 61±5%, P<0.05; nicotine: 76±6%, P<0.05), indicating that both L-AChRs and N-AChRs, were functionally compromised in these animals (Figure 4A and B). GABA-evoked PSCs were not affected (nra-2(tm1453): 103±17%; Figure 4C). Levamisole-induced PSCs in nra-2(ok1731); nra-4(hd127) double mutants were not further reduced than in single mutants, again indicating a function of NRA-2 and NRA-4 in the same pathway. Yet, nicotine-evoked PSCs were normal in these double mutants. Possibly, some nra-2 and nra-4 effects on L- and N-AChRs are allele specific, and such effects may be partly compensated in double mutants, for example, due to direct physical interactions of NRA-2 and NRA-4. Levamisole- and nicotine-induced PSCs in nra-2(ok1731) mutants were rescued by muscle-specific expression of NRA-2∷GFP (Figure 4A and B), confirming the cell-autonomous function of NRA-2. Figure 4.Whole-cell voltage-clamp analysis of muscle cells reveals altered nAChR function in nra-2 and nra-4 mutants. (A) Representative traces for levamisole- (top), nicotine- (middle) and ACh-evoked (bottom) muscle currents in wild-type animals and various mutants of nra-2, nra-4, L- and N-AChR subunits. (B) Normalized mean peak values of levamisole-, nicotine- and ACh-mediated muscle currents in wild-type animals and various nra-2 and nra-4 mutants, and nra-2(ok1731) animals rescued in muscle by NRA-2∷GFP expression. Only GFP-positive cells were patched. (C) Representative traces (left) and mean peak values (right) of GABA-mediated muscle currents were not altered in nra-2(tm1453) mutants, compared with wild type. (D) Normalized mean peak values of levamisole-, nicotine- and ACh-mediated muscle currents in wild-type animals, nra-2(tm1453 or ok1731) mutants as well as in mutants lacking the N-AChR (acr-16(ok789); left) or L-AChR (unc-38(x20); right), and respective double mutants. Displayed are means±s.e.m., statistically significant differences to the wild type are indicated (*P<0.05; **P<0.01; ***P<0.001), as well as the number of animals. Download figure Download PowerPoint Short-term ACh sensitivity of L- and possibly N-AChRs is increased in nra-2 mutants On the basis of agonist-evoked PSCs, both L- and N-AChRs are affected in nra-2 and nra-4 mutants. This is not seen in paralysis assays, as acr-16 mutants are not resistant to either agonist, in contrast to L-AChR mutants (Supplementary Figure 13), stressing differences between behavioural and electrophysiological phenotypes of L- versus N-AChR mutations. These could depend on the duration of agonist exposure, as L-AChRs desensitize much more slowly than N-AChRs. Surprisingly, PSCs in response to short-term ACh application in both nra-2 alleles, in nra-4(hd127) mutants and in several double-mutant combinations, were indistinguishable from the wild type (Figure 4A and B; Supplementary Figure 14). This was unexpected, as L- and N-AChRs are the only nAChRs contributing to cholinergic signalling at the NMJ (Richmond and Jorgensen, 1999; Francis et al, 2005; Touroutine et al, 2005). Our findings
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