An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse - PubMed (original) (raw)
An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse
Ruta B Almedom et al. EMBO J. 2009.
Abstract
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.
Figures
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
, 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.
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.
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.
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.
Figure 5
Optogenetic analysis of ACh transmission in cholinergic and nra-2 mutants using channelrhodopsin-2 (ChR2). (A) Whole-cell voltage-clamp was used to record photo-ePSCs in animals expressing ChR2 in cholinergic motor neurons (punc-17 promoter), in response to a 1000 ms photo-stimulus, as described earlier (Liewald et al, 2008). Representative peak and steady-state currents were compared in wild type, acr-16(ok789), unc-38(x20) and nra-2(ok1731) mutants. Duration of light stimulus is indicated by a bar. (B) Mean peak and steady-state photo-ePSCs, obtained using two different integrated transgenes, as indicated. Displayed are mean currents±s.e.m., statistically significant differences to the wild type are indicated (_t_-test; *P<0.05; ***P<0.001), as is the number of animals used.
Figure 6
Single-channel properties of the L-AChR in cell-attached patches of cultured embryonic muscle cells are altered in nra-2(ok1731) mutants. (A) Single-channel currents recorded from wild type and nra-2(ok1731) muscle cells in the presence of 1 μM (upper panel) and 50 μM (lower panel) ACh. Shown are representative traces (left) and open and closed time histograms (right). (B) Single-channel currents activated by 0.1 μM (upper panel) and 50 μM (lower panel) levamisole; representative traces (left) and open time histograms (right). (C) Frequency of channel openings in mutant and wild-type animals. Channel events were counted within the first minute of recording and plotted as events/s. Holding potential in all recordings was −100 mV. Displayed are means±s.d.
Figure 7
Contribution of essential and non-essential L-AChR subunits to cholinergic agonist sensitivity in nra-2(ok1731) mutants. (A, B) Paralysis assays (_n_=2–7; 30 animals each) in response to levamisole (A) and nicotine (B) of mutants in nra-2(ok1731), lev-8(x15), acr-8(ok1240) and unc-38(x20), and in double-mutant combinations as indicated. (C) Normalized mean peak values of ACh-, levamisole- and nicotine-induced muscle PSCs in wild-type animals, nra-2(ok1731) mutants, and mutants of the non-essential L-AChR α-subunits lev-8(x15) and acr-8(ok1240) as well as respective double mutants. Displayed are means±s.e.m., number of animals and significant differences to wild type (_t_-test; *P<0.05; **P<0.01; ***P<0.001) are indicated.
Figure 8
Individual L-AChR subunit levels at postsynaptic elements vary in nra-2 and nra-4 mutants reciprocally. (A, B, E) Synaptic expression of different L-AChR subunits was analysed by quantitative fluorescence microscopy. Endogenous, postsynaptic UNC-29, as well as the presynaptic UNC-17 (vAChT), were immunolabelled with specific 1° and different fluorescent 2° antibodies, then UNC-29 fluorescence was normalized to UNC-17 and compared in the indicated mutants. Also, transgenic animals expressing epitope-tagged LEV-1 (4 HA tags), UNC-38 (3 MYC tags), LEV-8 (3 HA tags) or ACR-8 (6 HA tags) were injected into the body cavity with fluorescent tag-specific antibodies. Size bar: 10 μm. (B) LEV-8 is non-uniformly expressed in the nervous system, as compared with UNC-38. Shown is expression of both subunits in the nerve ring, and the anterior and midbody ventral nerve cords (nc). (C, D, E) Fluorescence in the ventral cord was quantified (as linescans, followed by background correction) either in fixed animals (UNC-29, UNC-17), or in live animals after a recovery period of >6 h (during which excess antibody is cleared from the extracellular fluid by scavenger cells). Shown is mean fluorescence±s.e.m. (normalized to wild type in C, arbitrary units in D and E), number of animals and significant differences to wild type are indicated (_t_-test; *P<0.05; **P<0.01; ***P<0.001). (F) Model of NRA-2/NRA-4 function. Left, NRA-2/NRA-4 either influence the choice of particular subunits (indicated by different colours) to be assembled into pentameric nAChRs, or they determine to which extent pentamers of particular subunit composition are allowed to leave the ER (less favoured, as no obvious accumulation of NRA-2 was seen at ER exit sites). ACR-16 N-AChRs and L-AChRs, rarely incorporating ACR-8 subunits (yellow) are preferably formed. Right, In the nra-2 or nra-4 mutants, nAChRs of other composition are found, for example, containing ACR-8 or UNC-29 subunits more often. Depending on the allele, NRA-2 and NRA-4 proteins could either be completely absent, not bound to ER membranes and secreted, or of inverted topology.
References
- Changeux J, Edelstein S (2005) Nicotinic Acetylcholine Receptors. New York: Odile Jacob Publishing Corporation
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