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• W3048798742 abstract "•NACHO mediates functional assembly of transmembrane domains within the α7 nAChR•Two amino acids in the TM2 domain of α7 are critical for NACHO-mediated assembly•NACHO associates with the oligosaccharyltransferase machinery and with calnexin•Neuronal α7 function requires N-glycosylation and calnexin chaperone activity The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors. The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors. Nicotinic acetylcholine receptor (nAChR) ion channels are expressed throughout the brain and participate in synaptic transmission and plasticity (Gotti and Clementi, 2004Gotti C. Clementi F. Neuronal nicotinic receptors: from structure to pathology.Prog. Neurobiol. 2004; 74: 363-396Crossref PubMed Scopus (797) Google Scholar; Hogg et al., 2003Hogg R.C. Raggenbass M. Bertrand D. Nicotinic acetylcholine receptors: from structure to brain function.Rev. Physiol. Biochem. Pharmacol. 2003; 147: 1-46Crossref PubMed Scopus (395) Google Scholar; Le Novère et al., 2002Le Novère N. Corringer P.J. Changeux J.P. The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences.J. Neurobiol. 2002; 53: 447-456Crossref PubMed Scopus (348) Google Scholar; Lindstrom, 1997Lindstrom J. Nicotinic acetylcholine receptors in health and disease.Mol. Neurobiol. 1997; 15: 193-222Crossref PubMed Scopus (400) Google Scholar; Picciotto, 2003Picciotto M.R. Nicotine as a modulator of behavior: beyond the inverted U.Trends Pharmacol. Sci. 2003; 24: 493-499Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar; Role and Berg, 1996Role L.W. Berg D.K. Nicotinic receptors in the development and modulation of CNS synapses.Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). These receptors also mediate the behavioral and addictive properties of nicotine (Changeux, 2010Changeux J.P. Nicotine addiction and nicotinic receptors: lessons from genetically modified mice.Nat. Rev. Neurosci. 2010; 11: 389-401Crossref PubMed Scopus (338) Google Scholar). The neuronal nAChR family comprises nine α (α2–α10) and three β (β2–β4) subunits that form an array of pentameric channels. Acetylcholine (ACh) binds at subunit interfaces of the extracellular N-terminal region, which is followed by four transmembrane (TM) domains that form the channel pore. The most abundant nAChRs in brain are homomeric α7 receptors and heteromeric α4β2. Medicines that directly or indirectly modulate nAChRs are approved to treat a spectrum of disorders, including Alzheimer’s, Parkinson’s, and cardiovascular diseases. Particularly interesting is the α7 receptor, which is a compelling target for schizophrenia and autoimmune disorders (Dineley et al., 2015Dineley K.T. Pandya A.A. Yakel J.L. Nicotinic ACh receptors as therapeutic targets in CNS disorders.Trends Pharmacol. Sci. 2015; 36: 96-108Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar; Hurst et al., 2013Hurst R. Rollema H. Bertrand D. Nicotinic acetylcholine receptors: from basic science to therapeutics.Pharmacol. Ther. 2013; 137: 22-54Crossref PubMed Scopus (379) Google Scholar; Shi et al., 2009Shi F.D. Piao W.H. Kuo Y.P. Campagnolo D.I. Vollmer T.L. Lukas R.J. Nicotinic attenuation of central nervous system inflammation and autoimmunity.J. Immunol. 2009; 182: 1730-1739Crossref PubMed Scopus (76) Google Scholar). Whereas nAChRs are attractive therapeutic targets, progress is hampered because most nAChRs do not form functional channels in the cell lines used for drug discovery (Cooper and Millar, 1997Cooper S.T. Millar N.S. Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit.J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (131) Google Scholar; Kassner and Berg, 1997Kassner P.D. Berg D.K. Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells.J. Neurobiol. 1997; 33: 968-982Crossref PubMed Scopus (45) Google Scholar). This is curious, as other receptors in the Cys-loop superfamily, including GABAA, glycine, and 5-HT3 receptors robustly express functional channels in non-neuronal cell lines. Cell biological studies have determined that assembly of nAChRs is a complex and multi-step process that involves subunit biogenesis on rough endoplasmic reticulum (ER), oligomerization in the ER lumen, and transport through the Golgi to the cell surface (Green and Millar, 1995Green W.N. Millar N.S. Ion-channel assembly.Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (175) Google Scholar). Expression cloning in C. elegans determined that the single-pass TM protein resistance to inhibitors of cholinesterase-3 (RIC-3) is essential for worm nAChR assembly and function (Halevi et al., 2002Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors.EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar; Wang et al., 2009Wang Y. Yao Y. Tang X.Q. Wang Z.Z. Mouse RIC-3, an endoplasmic reticulum chaperone, promotes assembly of the alpha7 acetylcholine receptor through a cytoplasmic coiled-coil domain.J. Neurosci. 2009; 29: 12625-12635Crossref PubMed Scopus (32) Google Scholar). Whereas mammalian RIC-3 can modestly enhance α7 and some other mammalian nAChRs (Millar, 2008Millar N.S. RIC-3: a nicotinic acetylcholine receptor chaperone.Br. J. Pharmacol. 2008; 153: S177-S183Crossref PubMed Scopus (97) Google Scholar; Williams et al., 2005Williams M.E. Burton B. Urrutia A. Shcherbatko A. Chavez-Noriega L.E. Cohen C.J. Aiyar J. Ric-3 promotes functional expression of the nicotinic acetylcholine receptor α7 subunit in mammalian cells.J. Biol. Chem. 2005; 280: 1257-1263Crossref PubMed Scopus (129) Google Scholar), RIC-3 is neither necessary (Deshpande et al., 2020Deshpande A. Vinayakamoorthy R.M. Garg B.K. Thummapudi J.P. Oza G. Adhikari K. Agarwal A. Dalvi P. Iyer S. Thulasi Raman S. et al.Why does knocking out NACHO, but not RIC3, completely block expression of α7 nicotinic receptors in mouse brain?.Biomolecules. 2020; 10: 470Crossref Scopus (10) Google Scholar; Koperniak et al., 2013Koperniak T.M. Garg B.K. Boltax J. Loring R.H. Cell-specific effects on surface α7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein.J. Neurochem. 2013; 124: 300-309Crossref PubMed Scopus (18) Google Scholar) nor sufficient (Kuryatov et al., 2013Kuryatov A. Mukherjee J. Lindstrom J. Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional α7 AChRs.PLoS ONE. 2013; 8: e62246Crossref PubMed Scopus (20) Google Scholar) for robust α7 receptor assembly or function. Using a genome-wide cDNA screening strategy, we identified the four-pass TM protein NACHO, which can reconstitute homopentameric α7 nAChRs in cell lines and is essential for assembly of α7 nAChRs in neurons (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). NACHO is also important for assembly and function of several other nAChR combinations including the brain-enriched α4β2 and α6β2-containing receptors (Matta et al., 2017Matta J.A. Gu S. Davini W.B. Lord B. Siuda E.R. Harrington A.W. Bredt D.S. NACHO mediates nicotinic acetylcholine receptor function throughout the brain.Cell Rep. 2017; 19: 688-696Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, NACHO does not affect assembly of other Cys-loop receptors or any other channels tested (Matta et al., 2017Matta J.A. Gu S. Davini W.B. Lord B. Siuda E.R. Harrington A.W. Bredt D.S. NACHO mediates nicotinic acetylcholine receptor function throughout the brain.Cell Rep. 2017; 19: 688-696Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Several neurotransmitter receptor channels complexes contain auxiliary subunits (Huganir and Nicoll, 2013Huganir R.L. Nicoll R.A. AMPARs and synaptic plasticity: the last 25 years.Neuron. 2013; 80: 704-717Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar; Maher et al., 2017Maher M.P. Matta J.A. Gu S. Seierstad M. Bredt D.S. Getting a handle on neuropharmacology by targeting receptor-associated proteins.Neuron. 2017; 96: 989-1001Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar; Tomita and Castillo, 2012Tomita S. Castillo P.E. Neto1 and Neto2: auxiliary subunits that determine key properties of native kainate receptors.J. Physiol. 2012; 590: 2217-2223Crossref PubMed Scopus (48) Google Scholar). AMPA-type glutamate receptors co-purify with TARP subunits (Schwenk et al., 2012Schwenk J. Harmel N. Brechet A. Zolles G. Berkefeld H. Müller C.S. Bildl W. Baehrens D. Hüber B. Kulik A. et al.High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes.Neuron. 2012; 74: 621-633Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar; Tomita et al., 2004Tomita S. Fukata M. Nicoll R.A. Bredt D.S. Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.Science. 2004; 303: 1508-1511Crossref PubMed Scopus (187) Google Scholar), which mediate both AMPA receptor trafficking and gating (Tomita et al., 2005Tomita S. Adesnik H. Sekiguchi M. Zhang W. Wada K. Howe J.R. Nicoll R.A. Bredt D.S. Stargazin modulates AMPA receptor gating and trafficking by distinct domains.Nature. 2005; 435: 1052-1058Crossref PubMed Scopus (401) Google Scholar). In contrast, NACHO resides in the ER and does not stably associate with nAChRs. Instead, NACHO functions as nAChR-specific chaperone that enables subunit oligomerization and transit through the ER to the Golgi (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). How NACHO mediates nAChR assembly remains unknown. Here, we used molecular and neurobiological approaches to uncover mechanisms for NACHO effects on α7. A series of α7/5-HT3A chimeras determined that swapping the TM domains of 5-HT3A into α7 replaces and occludes effects of NACHO, which functions via the α7 extracellular N terminus as well as a pair of amino acids in TM domain 2. Proteomic studies show that NACHO co-purifies with ribophorin-1 and ribophorin-2, ER constitutes of the N-oligosaccharyl high mannose transferase complex (Kelleher and Gilmore, 2006Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (416) Google Scholar), and with calnexin, which is an ER chaperone that mediates folding of high mannose-containing polypeptides (Parodi, 2000Parodi A.J. Protein glucosylation and its role in protein folding.Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (535) Google Scholar). Indeed, NACHO-dependent assembly requires α7 N-glycosylation and calnexin interactions. Taken together, these studies provide mechanistic insights regarding NACHO-mediated nAChR assembly and suggest new pharmacological approaches to modulate this medically important neurotransmitter receptor. To identify α7 regions involved in NACHO-mediated surface trafficking, we constructed α7/5-HT3A chimeras (Figure 1A) containing extracellular HA tags to quantify receptor surface expression. Consistent with previously published studies (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), when transfected alone, α7 (C-terminal HA tag) surface staining was barely detectable, and co-expression with NACHO yielded robust HA labeling of non-permeabilized cells (Figures 1B, 1C, and S1A). In contrast, 5-HT3A (N-terminal HA tag) was robustly expressed on the cell surface, and co-expression with NACHO did not increase this (Figures 1B, 1C, and S1A). When the ligand-binding N terminus of α7 was replaced by the corresponding region of 5-HT3A (N-terminal HA tag), surface expression was not detected in the presence or absence of NACHO (Figures 1B, 1C, and S1A). In contrast, a chimera comprising the N terminus of α7 and the remainder 5-HT3A (C-terminal HA tag) showed strong surface labeling, and co-expression with NACHO did not further increase this (Figures 1B, 1C, and S1A). We conducted parallel electrophysiological studies with these chimeras. Confirming previously published studies (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), NACHO enabled ACh-induced whole-cell currents in α7-transfected cells and did not augment currents in 5-HT3A-transfected cells (Figures 1D and S1C). Consistent with our surface expression studies, the chimera containing the N terminus of α7 and the remainder 5-HT3A receptor showed robust ACh-evoked currents by itself, and these were not augmented by NACHO (Figures 1D and S1C). As previously published (Eiselé et al., 1993Eiselé J.L. Bertrand S. Galzi J.L. Devillers-Thiéry A. Changeux J.P. Bertrand D. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.Nature. 1993; 366: 479-483Crossref PubMed Scopus (360) Google Scholar), ACh-evoked currents from this chimera desensitized more slowly than those from wild-type α7, and these kinetics were not affected by NACHO (Figures 1D and S1D). Consistent with our surface expression experiments, the chimera containing the N terminus of 5-HT3A on α7 lacked agonist-evoked currents in the presence or absence of NACHO (Figures 1D and S1C). Taken together, these experiments show that the TM domains of α7 require NACHO for receptor functional expression and that an α7 chimera containing the TM domains from the 5-HT3A receptor is NACHO independent. We next used fluorescent α-bungarotoxin (α-Bgt-647), which binds at the interface of α7 subunits and therefore assesses assembly (Couturier et al., 1990Couturier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (817) Google Scholar; Schoepfer et al., 1990Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Consistent with previous studies (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), surface α-Bgt labeling of α7 required NACHO. In contrast and as previously published (Eiselé et al., 1993Eiselé J.L. Bertrand S. Galzi J.L. Devillers-Thiéry A. Changeux J.P. Bertrand D. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.Nature. 1993; 366: 479-483Crossref PubMed Scopus (360) Google Scholar), the chimera containing the N terminus of α7 on the 5-HT3A receptor (R227) exhibited high surface α-Bgt labeling that was not affected by NACHO (Figures 2B, 2C, and S1B). To further define α7 regions required for NACHO-mediated assembly and trafficking, we constructed additional chimeras containing the full α7 N-terminal domain and increasing TM portions of the channel (Figure 2A). When just 13 amino acids from the first TM domain of α7 were added to the 5-HT3A receptor chimera (P240), surface α-Bgt labeling was significantly decreased, and NACHO had minimal effect (Figures 2B, 2C, and S1B). When the full TM1 of α7 was added (G259), basal α-Bgt surface labeling was abolished, and NACHO modestly increased surface staining. Remarkably, adding just eight amino acids from TM2 (T267) enabled robust NACHO-mediated surface α-Bgt labeling to levels even higher than wild-type α7 (Figures 2B, 2C, and S1B). Taken together, these data suggest that amino acids from E260 to T267 in the second TM domain of α7 are required for NACHO-mediated assembly and trafficking. We further analyzed the eight amino acids from α7 TM2 needed for NACHO-mediated assembly and trafficking. Alignment of α7 and 5-HT3A receptors in this region identifies just four amino acid differences (Figure S2A); we generated point mutations for each and assessed NACHO-dependent assembly and trafficking. When K261 or I262 were switched to the corresponding amino acids from 5-HT3A receptor, NACHO retained robust effects on surface α-Bgt staining (Figure S2B). However, when L264 or G265 were changed to their 5-HT3A receptor counterparts, NACHO-mediated assembly and trafficking were significantly decreased (Figure S2B). We also found that simply mutating L264 or G265 on wild-type α7 abolished NACHO-mediated surface α-Bgt staining (Figure S2C), indicating that amino acids L264 and G265 in the second TM domain of α7 are critical for NACHO-mediated assembly and trafficking. RIC-3 protein is required for nAChR activity in C. elegans (Halevi et al., 2002Halevi S. McKay J. Palfreyman M. Yassin L. Eshel M. Jorgensen E. Treinin M. The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors.EMBO J. 2002; 21: 1012-1020Crossref PubMed Scopus (182) Google Scholar) and powerfully synergizes with NACHO for assembly and trafficking of human α7 (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). To compare α7 regions necessary for NACHO and RIC-3, we assessed their effects on the chimeras. As shown previously on wild-type α7 (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), RIC-3 alone had minimal effects and dramatically augmented NACHO-mediated α-Bgt staining (Figure 3B). However, when tested with chimeras containing some (T267) or all TM2 (P284) of α7 but lacking the large intracellular loop, RIC-3 lost its synergistic effect with NACHO (Figure 3). To test further whether RIC-3 effects with NACHO require the α7 large intracellular loop, we constructed a chimera containing only the final TM domain from 5-HT3A receptor (V466) (Figure 3A). Interestingly, RIC-3 retained its synergistic activity even though this chimera exhibited lower baseline NACHO-mediated surface expression (Figure 3B). Furthermore, swapping the large intracellular loop from α3 into α7 retained full NACHO activity but almost eliminated effects of RIC-3 (Figure S3). These results are consistent with previous studies showing that the large cytoplasmic region of α7 is required for significant RIC-3 effects and that RIC-3 has modest effects on α3-containing nAChRs (Castillo et al., 2006Castillo M. Mulet J. Gutiérrez L.M. Ortiz J.A. Castelán F. Gerber S. Sala S. Sala F. Criado M. Role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors.J. Mol. Neurosci. 2006; 30: 153-156Crossref PubMed Scopus (19) Google Scholar; Millar and Harkness, 2008Millar N.S. Harkness P.C. Assembly and trafficking of nicotinic acetylcholine receptors (Review).Mol. Membr. Biol. 2008; 25: 279-292Crossref PubMed Scopus (108) Google Scholar). Nicotine and other cholinergic ligands stabilize assembled nAChRs, and this likely contributes to nicotine addiction (Lester et al., 2009Lester H.A. Xiao C. Srinivasan R. Son C.D. Miwa J. Pantoja R. Banghart M.R. Dougherty D.A. Goate A.M. Wang J.C. Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery.AAPS J. 2009; 11: 167-177Crossref PubMed Scopus (140) Google Scholar; Schwartz and Kellar, 1983Schwartz R.D. Kellar K.J. Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo.Science. 1983; 220: 214-216Crossref PubMed Scopus (420) Google Scholar). Accordingly, we found that 24 h preincubation with the nicotinic antagonist, methyllycaconitine (MLA), which is a pharmacological chaperone of α7 (Kuryatov et al., 2013Kuryatov A. Mukherjee J. Lindstrom J. Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional α7 AChRs.PLoS ONE. 2013; 8: e62246Crossref PubMed Scopus (20) Google Scholar), augmented α7 assembly and trafficking in a NACHO-dependent fashion (Figure S4A). To test for interactions of ligand binding with NACHO- and RIC-3-mediated surface trafficking, we mutated a critical residue, tryptophan (W) 171 within the ACh-binding pocket of α7 (Hansen et al., 2005Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations.EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (575) Google Scholar). As expected, this mutant disrupted ACh-evoked calcium influx from α7 in the presence or absence of NACHO and/or RIC-3 (Figure S4B). In contrast, NACHO and RIC-3 worked synergistically to enable surface expression of this mutant, indicating that ligand binding is not required for NACHO or RIC-3 effects (Figures S4C and S4D). We next asked whether the α7 N terminus and first two TM domains would be required for NACHO-mediated assembly in the context of a nAChR that is not regulated by NACHO. As NACHO promotes assembly of α3-, α4-, and α6-containing nAChRs (Matta et al., 2017Matta J.A. Gu S. Davini W.B. Lord B. Siuda E.R. Harrington A.W. Bredt D.S. NACHO mediates nicotinic acetylcholine receptor function throughout the brain.Cell Rep. 2017; 19: 688-696Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), we assessed α9, which is somewhat more distantly related. In oocytes but not in HEK cells, α9 can form functional homopentameric receptors (Elgoyhen et al., 1994Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (747) Google Scholar). We found that NACHO in the presence or absence of MLA had no effect on α9 assembly (Figures 4B, 4C, and S4E). We therefore constructed a set of α7/α9 chimeras (Figure 4A). When just the N terminus of α7 was swapped into α9, NACHO-mediated assembly was detectable in the presence of MLA (Figures 4B and 4C). Another chimera containing the first and second TM domains of α7 also showed significant NACHO-mediated assembly in the presence of MLA (Figures 4B and 4C). Taken together, these chimera studies show that the TM domains of α7 or α9 are incompetent for receptor assembly and that NACHO engagement with the α7 extracellular N-terminal region can rectify this for either receptor. We next explored whether NACHO-mediated assembly in neurons also requires the first 267 amino acids from α7. We first transduced cultured cortical neurons from wild-type or NACHO-knockout (KO) mice with lentivirus encoding HA-tagged α7. Surface α-Bgt and anti-HA staining showed assembly and trafficking of α7 in wild-type neurons but not in NACHO-KO neurons (Figure 5A). The lack of surface α7 expression in NACHO-KO neurons was not due to the lack of total protein expression of α7 (Figure 5A). Instead, total anti-HA staining in permeabilized cultures showed that α7 distributed throughout the dendritic arbors in wild-type neurons but was restricted to the neuronal soma in NACHO KO. MAP2 staining showed that wild-type and NACHO-KO neurons had similar dendritic morphologies (Figure 5A). To determine whether amino acids 1–267 of α7 are required for NACHO-mediated assembly, we transduced cultured neurons with lentiviruses encoding α7 or α7/5-HT3A chimeras. Consistent with our staining experiments in HEK293T cells, the chimera containing the N terminus of α7 on the 5-HT3A receptor (R227) exhibited α-Bgt labeling that was not affected by NACHO, whereas the chimera containing the first 267 amino acids from α7 (T267) specifically showed NACHO-dependent α-Bgt labeling (Figure 5B). These data validate that NACHO-mediated assembly in neurons requires TM domains 1 and 2 of α7. Previous studies found that NACHO does not associate stably with α7 (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). To identify protein partners of NACHO, we prosecuted a proteomics approach. To best maintain protein-protein interactions, we solubilized mouse forebrain membranes with a calixarene-based detergent (Rosati et al., 2015Rosati A. Basile A. D’Auria R. d’Avenia M. De Marco M. Falco A. Festa M. Guerriero L. Iorio V. Parente R. et al.BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages.Nat. Commun. 2015; 6: 8695Crossref PubMed Scopus (65) Google Scholar). Solubilized proteins were immunoprecipitated with anti-NACHO antibody or control IgG, and associated proteins were identified by mass spectrometry. Parallel experiments from NACHO-KO mice were used to assess specificity. Interacting proteins were those specifically identified in replicate anti-NACHO immunoprecipitations and not in IgG control immunoprecipitations from wild-type but not NACHO-KO protein homogenates. Spectral counts of three proteins were detected at levels at least 50% of those found for NACHO itself (Table S1). Interestingly, these three proteins—ribophorin-1, ribophorin-2, and calnexin—are residents of the ER involved in membrane protein biogenesis and folding. Ribophorin-1 and ribophorin-2 are subunits of the oligosaccharyltransferase (OST) complex that links high mannose sugars to asparagine (N) residues of nascent polypeptides (Kelleher and Gilmore, 2006Kelleher D.J. Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase.Glycobiology. 2006; 16: 47R-62RCrossref PubMed Scopus (416) Google Scholar), and calnexin is a protein chaperone (Parodi, 2000Parodi A.J. Protein glucosylation and its role in protein folding.Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (535) Google Scholar) that retains unfolded N-linked glycoproteins. N-glycosylation is required for maturation of many receptors and ion channels (Parodi, 2000Parodi A.J. Protein glucosylation and its role in protein folding.Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (535) Google Scholar), including nACh receptors (Chen et al., 1998Chen D. Dang H. Patrick J.W. Contributions of N-linked glycosylation to the expression of a functional alpha7-nicotinic receptor in Xenopus oocytes.J. Neurochem. 1998; 70: 349-357Crossref PubMed Scopus (54) Google Scholar). We did not detect α7 or other nAChRs, and this fits with our previous finding showing that α7 and NACHO do not interact stably (Gu et al., 2016Gu S. Matta J.A. Lord B. Harrington A.W. Sutton S.W. Davini W.B. Bredt D.S. Brain α7 nicotinic acetylcholine receptor assembly requires NACHO.Neuron. 2016; 89: 948-955Abstract Fu" @default.
• W3048798742 created "2020-08-18" @default.
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• W3048798742 date "2020-08-01" @default.
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• W3048798742 title "NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly" @default.
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