- Email: [email protected]
Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO
Report
Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO Highlights d
d
d
d
Genomic screening identifies NACHO as a mediator of a7 nAChR functional expression NACHO promotes folding and surface expression of a7 nAChRs
Authors Shenyan Gu, Jose A. Matta, Brian Lord, Anthony W. Harrington, Steven W. Sutton, Weston B. Davini, David S. Bredt
NACHO is a neuronal ER-resident protein that enhances a7 nAChR biogenesis
Correspondence
Alpha7 nAChR assembly and function in brain require NACHO
In Brief
[email protected]
Gu et al. used high-throughput screening to identify NACHO, a unique mediator of a7 nACh receptor assembly, biogenesis, and membrane insertion. Complete absence of functional a7 receptors in NACHO knockout mice establishes the essential role for this novel mechanism.
Gu et al., 2016, Neuron 89, 1–8 March 2, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2016.01.018
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
Neuron
Report Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO Shenyan Gu,1,2 Jose A. Matta,1,2 Brian Lord,1 Anthony W. Harrington,1 Steven W. Sutton,1 Weston B. Davini,1 and David S. Bredt1,* 1Neuroscience
Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2016.01.018 2Co-first
SUMMARY
Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing a7-type nAChR signaling improves symptoms in Alzheimer’s disease and schizophrenia. The pharmaceutical study of a7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie a7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of a7 receptors. NACHO promotes a7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts a7 receptor assembly and abolishes a7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors. INTRODUCTION Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate diverse physiological responses including pain processing and cognitive functions (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996). The a7 subtype is especially important, as this receptor not only participates in learning and memory (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996), but also underlies a cholinergic anti-inflammatory pathway relevant to autoimmune disorders (Wang et al., 2003) and stimulates cell proliferation and angiogenesis in lung and pancreatic cancers (Schuller, 2009). Upregulation of nAChRs represents a therapeutic goal and likely contributes to the pharmacology (Lester et al., 2009; Schwartz and Kellar, 1983) of cholinergic medicines in Alzheimer’s disease and schizophrenia (Dineley
et al., 2015; Hurst et al., 2013). Achieving this goal requires elucidation of fundamental mechanisms that control nAChR biogenesis. Nicotinic AChRs are members of the Cys-loop receptor family that include glycine, GABA-A, and 5-HT3 receptors, which all contain five subunits arranged in a ring surrounding the ion channel pore (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997). Assembly of nAChRs is a slow and tightly regulated process (Green and Millar, 1995) in which subunits first undergo a series of post-translational modifications followed by subunit-subunit interactions to form functional pentamers. Several general protein chaperones including BiP, ERp57, and calnexin modulate the trafficking and maturation of nAChRs (reviewed in Colombo et al., 2013). These ERresident proteins modulate biogenesis of numerous transmembrane proteins, and their precise roles in assembly of nAChR remain uncertain. Alpha7 nACh receptors are calcium-permeable homo-pentamers (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996), and they do not properly oligomerize or functionally express in almost all cell lines (Cooper and Millar, 1997; Kassner and Berg, 1997; Koperniak et al., 2013; Kuryatov et al., 2013). In C. elegans, resistance to inhibitors of cholinesterase-3 (Ric3) is required for nAChR activity (Nguyen et al., 1995). Whereas the mammalian Ric-3 homolog modestly increases a7 activity and has diverse effects on other co-expressed nACh and 5-HT3 receptors (Halevi et al., 2003; Millar, 2008), it is neither necessary (Koperniak et al., 2013) nor sufficient (Kuryatov et al., 2013) for efficient assembly of mammalian a7 receptors. Although some neurotransmitter receptors have auxiliary subunits that control receptor trafficking and channel gating (Jackson and Nicoll, 2011; Tomita and Castillo, 2012), no essential assembly proteins have previously been identified for a mammalian neurotransmitter receptor. RESULTS High-Throughput Screening Identifies NACHO as a Mediator of a7 Functional Expression To identify novel regulators of nAChR assembly and function, we utilized human embryonic kidney 293T (HEK) cells for highthroughput screening. In HEK cells transfected with a7 alone, ACh-evoked calcium influx, quantified with a fluorescence imaging plate reader (FLIPR), was undetectable, and application of Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 1
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
ACh + the a7 potentiator PNU-120596 (Hurst et al., 2005) produced a small calcium signal (Figure S1A). For FLIPR screening in a 384-well plate format, ACh (25 mM) and ACh plus PNU-120596 (10 mM) were sequentially applied to HEK cells in wells that had been individually co-transfected with a7 plus one of 3,880 clones from a cDNA library encoding almost all human transmembrane and secreted proteins (Origene). The single library clone that most profoundly augmented ACh + PNU-120596-induced responses (Figure S1A) derived from TMEM35, also termed unknown factor-1 (TUF-1), which encodes a small (167 amino acid) uncharacterized four-pass transmembrane protein (Figures S2A and S2B), whose expression in adrenal zona glomerulosa cells is increased by sodium depletion (Tran et al., 2010). We named this novel nAChR regulator (NACHO). To evaluate the effect of NACHO on a7 receptors, we performed electrophysiological experiments. Consistent with published studies (Cooper and Millar, 1997; Williams et al., 2005), we could not detect ACh-evoked currents in HEK cells transfected with a7 alone. Strikingly, co-transfection of NACHO with a7 yielded large, rapidly desensitizing ACh-evoked currents (Figures 1A and 1B). NACHO also significantly increased ACh-evoked currents in HEK cells co-transfected with a4 + b2 nAChR subunits (Figures 1A and 1B), which form hetero-pentameric receptors (Gotti and Clementi, 2004). In contrast, NACHO had no significant effect on glutamate- or 2-methyl-5-HT-evoked currents in cells co-transfected with NACHO and GluA1 or 5-HT3A/B receptors, respectively (Figures 1A and 1B). Furthermore, NACHO did not affect other ionotropic receptors tested including NMDA receptors, kainate receptors, TrpV1, and TrpM8 (Figure S1B). We asked whether NACHO increases surface expression of nAChR proteins. Therefore, we engineered in extracellular HA-tags that do not interfere with channel function and enable immunofluorescent detection of surface receptors. In HEK cells transfected with such a HA-tagged a7 construct alone, no labeling was detected following incubation of unpermeabilized cells with an anti-HA antibody (Figures 1C and 1D). Remarkably, cotransfection of NACHO yielded strong surface labeling for a7 (Figures 1C and 1D). We next evaluated the effect of NACHO on surface expression of HA-tagged a4b2, GluA1, and 5-HT3A/ B proteins. Consistent with our electrophysiological data, NACHO markedly increased a4b2 surface protein levels and had no effect on GluA1 or 5-HT3A/B (Figures 1C and 1D). NACHO is distantly related to only one human gene, ZMYM6NB, which also encodes a small, multi-pass transmembrane protein. We found that ZMYM6NB does not enhance the functional expression of a7 in co-transfected HEK cells (Figures S3A and S3B). A single potential ortholog of NACHO is present in the genomes of all vertebrates and some lower organisms. The NACHO homolog in Drosophila melanogaster (DNACHO) shares just 37% identity with human NACHO, yet it fully augments both functional and surface expression of co-transfected human a7 (Figures 1B and 1D). NACHO Promotes Assembly of a7 nAChR and Works Synergistically with Ric-3 We compared functional effects of NACHO with Ric-3, which is essential for nAChR responses in C. elegans (Nguyen et al., 1995) but whose mammalian homolog has mixed effects on a7, 2 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
a4b2, and 5-HT3 receptors (Halevi et al., 2003; Millar, 2008). In transfected cell lines, mammalian Ric-3 only modestly enhances a7 activity (Kuryatov et al., 2013). Endogenous Ric-3 is absent from hippocampal dentate gyrus neurons that are enriched in a7 (Halevi et al., 2003). Recent studies showed that complete knockdown of Ric-3 does not diminish a7 function in rat pituitary cells (Koperniak et al., 2013) demonstrating that endogenous Ric-3 is not essential. In whole-cell recordings from a7 co-transfected HEK cells, NACHO induces much larger ACh-evoked currents than does Ric-3 (Figures 2A and 2B). Interestingly, when both NACHO and Ric-3 are co-transfected, they synergistically increase ACh-evoked currents from a7 receptors, suggesting that they work through distinct mechanisms (Figures 2A and 2B). Assembly of a7 receptors can be quantified with a-bungarotoxin (a-Bgt), a snake venom component, which only binds to properly-folded a7 receptors (Couturier et al., 1990; Schoepfer et al., 1990). We used a-Bgt and a7 surface labeling, to compare effects of NACHO and Ric-3 on folding and trafficking of cotransfected a7-HA. In HEK cells transfected with a7-HA alone, neither surface a7 protein (purple) nor fluorescent a-Bgt binding before (red) or after (green) cell permeabilization could be detected (Figure 2C). In cells co-transfected with Ric-3 and a7-HA, a-Bgt binding and surface a7 labeling are also below our detection limits (Figure 2C). In striking contrast, co-transfection of a7-HA with NACHO yields abundant a-Bgt binding and a7 protein expression on the surface of unpermeabilized cells (Figure 2C). Following permeabilization of these a7 + NACHO co-transfectants, additional a-Bgt binding occurs (Figure 2C), which indicates the presence of folded intracellular a7 receptors that are not apparent without permeabilization (Figure 2C0 ). Cotransfection of NACHO and Ric-3 together with a7-HA further increases both surface a-Bgt binding and surface a7 protein levels and has no further effect on intracellular a-Bgt binding sites (Figure 2C). Immunofluorescent labeling for a7 following permeabilization shows that total a7 levels are similar in all transfection conditions (Figure 2C, cyan). NACHO Is a Neuronal ER-Resident Protein that Enhances a7 Biogenesis NACHO mRNA is uniquely enriched in brain (GTEx Project [Consortium, 2015]) and is discretely expressed in specific regions including hippocampus, cerebral cortex, and olfactory bulb (Figure S4A). Immunoblotting of rat hippocampal membranes or NACHO-transfected HEK cells with a NACHO antibody detects a protein band migrating at 18 kDa (Figure S4B), consistent with the predicted size of NACHO protein. This band is not present in rat liver or untransfected HEK cells. Importantly, this band is present (Figure S4B) in two neuroendocrine cell lines (PC12 and GH4C1) that can functionally express transfected a7 (Cooper and Millar, 1997; Koperniak et al., 2013). Immunofluorescent labeling of cultured hippocampal cells shows that NACHO occurs in neurons (identified with MAP2) and is not present in astrocytes (identified with GFAP) (Figure 3A). Higher-power magnification shows that NACHO co-localizes with an ER-resident protein disulfide isomerase (PDI) (Figure 3B). This ER localization of NACHO may be mediated through its extreme C-terminal tail (Figure S2A), which contains a canonical recognition sequence (KxKxx) that typically binds to b’-COP
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
B
C
D
Figure 1. NACHO Promotes Surface Expression of Functional nAChRs (A) Representative whole-cell recordings from HEK cells co-transfected with GFP and cDNAs encoding a7 /+NACHO; a4b2 /+NACHO; GluA1 /+NACHO; or 5-HT3A/B /+NACHO. (B) Summary graphs of agonist-evoked peak currents in HEK cells transfected as indicated (mean ± SEM). p values of comparisons ± NACHO: a7 p = 0.015, a4b2 p = 0.002, GluA1 p = 0.60, 5-HT3A/B p = 0.23. DNACHO represents the Drosophila melanogaster homolog (p = 0.028 ± DNACHO). (C) HEK cells were transfected as in (A) but with extracellular, HA-tagged receptors and were fluorescently labeled without permeabilization with anti-HA. (D) Quantification of surface labeling normalized to GFP (mean ± SEM, n = 5), p < 0.0001 ± NACHO in a7 or a4b2.
subunits for Golgi-to-ER retrieval (Jackson et al., 2012). Surface biotinylation on cultured hippocampal neurons did not detect NACHO protein on the surface of neurons (Figure 3C), consistent with NACHO being an intracellular (ER) protein. In a7-transfected HEK cells with or without Ric-3, all a7 protein is sensitive to Endo H glycosidase (Figure 3D), indicating that a7 in these cells does not progress properly from ER through Golgi. By contrast, co-transfection with NACHO generates an Endo
H-resistant population of a7 (Figure 3D), implying that NACHO enables a7 maturation. Ric-3 further enhances the effect of NACHO on a7 maturation (Figure 3D). Whereas NACHO promotes a7 assembly and biogenesis, we found that NACHO does not alter the desensitization or deactivation kinetics of either a7 (Figures S5A and S5B) or a4b2 (Figures S5D and S5E). We also investigated recovery from desensitization, which evaluates response to ACh at increasing intervals following a Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 3
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
C
B
C’
Figure 2. NACHO Mediates Assembly of a7 nAChR and Synergizes with Ric-3 (A) Whole-cell recordings from HEK cells transfected with cDNAs as indicated. (B) Quantification of ACh-evoked peak currents (mean ± SEM). Student’s t test p values: NACHO versus Ric-3 = 0.002; NACHO + Ric-3 versus NACHO = 0.0034. (C) Fluorescent labeling of HEK cells transfected as indicated. Surface staining of non-permeabilized cells used anti-HA (purple) and Bgt-555 (red). Following permeabilization, cells were labeled with Bgt-488 (green) to identify folded intracellular a7 and finally with anti-a7 (cyan) to quantify total receptor expression. (C0 ) As control, cells transfected with a7 + NACHO + Ric-3 were processed without permeabilization, which confirmed lack of labeling for Bgt-488 (green) after Bgt-555 (red).
desensitizing pulse of ACh. Again, NACHO does not affect recovery from desensitization for either a7 (Figure S5C) or a4b2 (Figure S5F). Furthermore, we performed immunoprecipitation experiments to assess whether NACHO and a7 tightly interact in hippocampal tissue. Because no available antibodies are suitable for western blotting of a7 in rodent brain (Moser et al., 2007), we detected a7 by [125I]a-Bgt binding. These experiments showed that NACHO immunoprecipitation does not bring down [125I]a-Bgt binding sites (Figure 3E). Conversely, a7 immunoprecipitation does not bring down NACHO protein. These results show that NACHO and a7 do not form a stable complex. Taken together, these data indicate that NACHO is not a surface-expressed a7 auxiliary subunit but rather is a neuronal, ER-localized chaperone for a7. Alpha7 Receptor Function in Brain Requires NACHO To evaluate functions for neuronal NACHO, we first identified an shRNA construct that effectively suppresses NACHO expres4 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
sion in hippocampal neurons (Figure 4B, purple). We quantified a7-mediated sustained currents in hippocampal neurons by applying ACh (1 mM) in the presence of PNU-120596 (1 mM), an a7 potentiator, that blocks the receptor desensitization. Remarkably, our shRNA-mediated knockdown of NACHO abolished the a7-mediated currents (Figure 4A). For a4b2, we quantified currents evoked by ACh (1 mM) that are sensitive to 10 mM dihydro-b-erythroidine (DhbE). We found that NACHO knockdown significantly decreases but does not eliminate these a4b2-mediated currents (Figure S6D), which fits our data in HEK cells that NACHO augments a4b2 currents (Figure 1B). These effects were specific as NACHO knockdown did not affect AMPA receptor-mediated currents (Figure 4A). Transfection of hippocampal neurons with an HA-tagged a7 yields abundant cell surface a7 and surface a-Bgt binding sites (Figure 4B, red). By contrast, HA-tagged a7 in neurons lacking NACHO are not present on the cell surface and do not form a-Bgt binding sites (Figure 4B).
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
B
C
E
D
Figure 3. NACHO Is an Intracellular Neuronal Protein that Promotes a7 Biogenesis (A) Immunofluorescent labeling of cultured rat hippocampal neurons shows that NACHO (red) occurs in MAP2-containing neurons (green) and does not colocalize with the astrocyte marker GFAP (purple). (B) Higher-power immunofluorescent labeling of hippocampal neurons shows co-localization of NACHO with the ER marker PDI (green). (C) Surface biotinylation of hippocampal neurons followed by immunoblotting shows that NACHO is exclusively intracellular. GluA1 and calnexin serve as references for cell surface and cytoplasmic proteins, respectively. Surface proteins were concentrated 5-fold (mean ± SEM, n = 3). (D) Glycosidase digestion of membranes from transfected HEK cells shows that NACHO promotes complex glycosylation of a7 as evidenced by resistance to Endo H treatment. Alone, Ric-3 has no effect on a7 glycosylation, but Ric-3 synergizes with NACHO. (E) Immunoprecipitation experiments show that anti-NACHO antibody does not pull down assembled a7, as detected by [125I]a-Bgt (mean ± SEM, n = 3), and that anti-a7 antibody does not pull down NACHO protein, as detected by immunoblotting.
We derived NACHO knockout mice (TMEM35tm1(KOMP)Vlcg) from the trans-NIH knockout mouse project (KOMP) repository (UC Davis). These knockout mice are viable, and they entirely lack NACHO protein and mRNA in brain (Figures S6A and S6B), whereas a7 mRNA is unaffected (Figure S6B). Electrophysiological recordings from acutely isolated hippocampal neurons showed complete absence of a7-mediated currents in NACHO knockouts. This deficit is specific, as AMPA receptors remain intact (Figure 4C). Furthermore, [125I]a-Bgt autoradiography shows that properly folded a7 receptors are undetectable in NACHO knockout brain (Figures 4D and S6C). DISCUSSION This study identifies NACHO as an essential factor for folding, maturation, and membrane insertion of a7. Our high-throughput screen of all predicted human transmembrane proteins indicates that NACHO has, by far, the greatest capacity to enhance assembly and function of a7. We find that the Drosophila NACHO ortholog effectively reconstitutes human a7 folding and function, which implies that the NACHO mechanism is
phylogenetically conserved. Most importantly, genetic deletion of NACHO abolishes functional a7 receptors throughout the brain. Whereas NACHO is required for a7 channel activity in brain, our heterologous cell and neuronal transfection experiments show that NACHO does not alter total a7 protein levels. The disposition of a7 protein in NACHO knockout mice is unknown because, unfortunately, no antibodies are suitable for immunostaining or immunoblotting of a7 in mouse brain (Moser et al., 2007). However, the complete loss of [125I]a-Bgt binding sites in NACHO knockout brain demonstrates absence of folded a7 pentamers. Cell biological properties distinguish NACHO from previously described neurotransmitter receptor partner proteins. Certain glutamate-gated ion channels, including AMPA and kainate receptors, also require neuronal accessory proteins (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). Best studied are transmembrane AMPA receptor regulatory proteins (TARPs), which, by analogy to NACHO, enhance surface trafficking of AMPA receptors (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). However, TARPs are not essential for AMPA receptor folding or tetramer assembly. Instead, TARPs are auxiliary Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 5
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
C
B
D
Figure 4. NACHO Is Required for Folding and Function of a7 Receptors in Brain (A) NACHO knockdown in rat hippocampal neurons selectively abolishes a7 channel activity. Whole-cell recordings of hippocampal neurons show that transfection with NACHO shRNA (red)—but not control shRNA (black)—eliminates ACh + PNU-120596 (PNU)-evoked currents (mean ± SEM, p < 0.0001). As control, NACHO knockdown does not affect currents evoked by Glu + cyclothiazide (CTZ) (mean ± SEM, p = 0.4562). (B) Fluorescent labeling shows that in a transfected neuron (green), NACHO shRNA effectively reduces endogenous NACHO protein (purple) and abolishes a-Bgt binding (red) and surface expression of co-transfected a7-HA (cyan). (C and D) NACHO knockout mice lack functional or folded a7 receptors. (C) Whole-cell recordings show that NACHO knockout (KO) mice (red) as compared to wild-type (black) do not display ACh + PNU-evoked currents but show normal responses to Glu + CTZ. Summary graphs for ACh + PNU-evoked (mean ± SEM, p < 0.0001) and Glu + CTZ-evoked currents in wild-type and NACHO knockout mice (mean ± SEM, p = 0.9817). (D) [125I]a-Bgt autoradiography on sagittal brain slices from wild-type and knockout mice shows that NACHO is essential for a7 receptor folding. CX, cortex; HP, hippocampus; SC, superior colliculus; IC, inferior colliculus; HY, hypothalamus.
subunits of AMPA receptors and associate with surface channels to control gating and pharmacology (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). By contrast, NACHO is fundamentally required for folding and assembly of a7 receptors and is the first essential, client-specific chaperone identified for a mammalian neurotransmitter receptor. The neuronal nAChR family comprises nine a and three b subunits, and all receptors other than a7-10 contain heteromeric subunits (Gotti and Clementi, 2004; Le Nove`re et al., 2002). Certain biologically important AChR subtypes, including a6b2containing receptors, cannot be functionally expressed in recombinant systems (Kuryatov et al., 2000). We also find that NACHO and Ric-3 are insufficient to promote functional expres6 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
sion of a6b2-containing nAChR (data not shown), which suggests that additional subtype-specific chaperones likely exist for these nAChR subtypes. Furthermore, selective chaperones may well mediate assembly of other neurotransmitter receptors. Neurotransmitter receptor auxiliary subunits and regulatory proteins have mostly been discovered by genetic screens (Nguyen et al., 1995; Zheng et al., 2004) and biochemical techniques (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). Whereas these approaches have been applied to nAChRs, they did not identify NACHO. Our discovery of NACHO took advantage of a high-throughput screening strategy enabled by the recent availability of genome-wide cDNA libraries. Our new approach complements existing strategies and should help
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
identify assembly proteins and auxiliary subunits for numerous other receptors and ion channels. Genetic, biochemical, and pharmacological studies validate a7 as a therapeutic target for treating Alzheimer’s disease and schizophrenia (Dineley et al., 2015; Freedman, 2014; Gotti and Clementi, 2004). Challenges in reliably reconstituting functional a7 in cells lines have impeded pharmacological studies. Coexpression of NACHO with a7 solves this issue and will facilitate drug discovery. The chaperone function of NACHO may also represent a valuable drug target. Nicotine itself promotes maturation of several types of nAChRs (Schwartz and Kellar, 1983), and this likely contributes to nicotine’s inadvertent beneficial effects in certain cognitive and neurodegenerative conditions (Lester et al., 2009). Enhancing NACHO effects on nAChR assembly could provide a medical target devoid of nicotine’s addictive and toxic properties. EXPERIMENTAL PROCEDURES 2+
FLIPR Assay for Ca Influx in HEK Cells High-throughput FLIPR assays were performed using HEK cells in 384-well plates. Briefly, cells were seeded at 1 3 104 cells per well and transfected 4 hr after seeding with Fugene HD. After 2 days of incubation, cells were washed three times with assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES [pH7.4]) and loaded with Calcium5 dye (Molecular Device) and 1.25 mM probenecid for 1 hr at RT. After three washes, the plates were placed in the FLIPR stage. After establishing baseline, 25 mM acetylcholine was applied for a 1.5 min recording, then 25 mM acetylcholine plus 10 mM PNU120596 were applied for another 2.5 min recording. a-Bgt and Immunofluorescent Staining For surface staining, cells were incubated with 1 mg/ml a-Bgt or primary antibodies in culture medium for 30 min at 37 C. Cells were then washed and fixed with 4% paraformaldehyde. As indicated, cells were permeabilized with 0.3% Triton X-100 and incubated with antibodies or a-Bgt to probe intracellular components. Following incubation with secondary antibodies, cells were stained with DAPI for 5 min. Images were captured using a Zeiss M2 Imager or Zeiss LSM 700 confocal microscope. [125I] a-BTX Autoradiography [125I]a-BTX Autoradiography was performed as described (Orr-Urtreger et al., 1997). Non-specific binding was assessed by inclusion of 1 mM unlabeled a-BTX. At the end of the incubation, sections were washed four times and dried. Images were captured with b-Imager DFine (Biospacelab). Electrophysiology Forty-eight hours post-transfection, transfected cells were transferred to the recording chamber submerged in extracellular solution composed of 137 mM NaCl, 10 mM HEPES, 5 mM glucose, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2 (pH 7.4), and 300 mM mOsm. For cultured neurons, synaptic activity was blocked with TTX (0.5 mM), NBQX (10 mM; except when measuring whole-cell AMPA currents), and d-APV (50 mM). Whole-cell recordings used intracellular solution composed of 140 mM potassium gluconate, 10 mM HEPES, 4 mM Mg-ATP, 0.4 mM Na-GTP, and 0.6 mM EGTA (pH 7.3). Ultrafast acetylcholine was applied onto HEK cells using a piezo-driven perfusion system and theta glass (Siskiyou). Fast perfusion onto neurons used a Perfusion Fast-Step System (Warner Instruments). Acute hippocampal neurons were obtained from 7-week-old male mice. Horizontal slices, 300 mm thick, were obtained using a VT1200S microtome (Leica Biosystems). Slices were cut in ice-cold solution composed of 150 mM sucrose, 50 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 7 mM MgSO4, 2.5 mM KCl, 1.25 mM Na3PO4, and 0.5 mM CaCl2 equilibrated with 95% O2 and 5% CO2. Hippocampi were isolated and transferred to Hiberna-
teA (no calcium, BrainBits) solution containing 1 mg/ml papain (Worthington) and 0.5 mM Glutamax (Life Technologies) and digested at 37 C for 30 min. Slices were gently triturated with fire-polished Pasteur pipettes. The cell suspension was then plated onto coverslips and isolated neurons were used for whole-cell patch-clamp recordings. All dissociated cell recordings were done at room temperature and membrane potentials were held at 60 mV. Recordings used an AxoPatch 200B amplifier (Axon Instruments). For transfected HEK cells, signals were filtered at 10 kHz and digitized at 50 kHz. For neurons, signals were filtered at 2 kHz and digitized at 10 kHz. Data acquisition and online analysis were done using pClamp 9 (Axon Instruments). For recovery from desensitization, an initial desensitizing pulse of ACh was followed by a second pulse of ACh at varying time intervals. At each time interval, recovery from desensitization was expressed as the fraction of the current peak amplitude of the second pulse to the first pulse. Current decay kinetics and recovery from desensitization were fitted with double exponential functions and expressed as weighted decay time constants. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and six figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.neuron.2016.01.018. AUTHOR CONTRIBUTIONS D.S.B. supervised the project. J.A.M. and S.G. performed electrophysiological studies and data analyses. B.L. performed autoradiography. A.W.H. and S.G. performed immunofluorescent staining. W.B.D. and S.G. performed hippocampal neuron culturing and transfections. S.W.S. contributed to knockout animal derivation. S.G. performed all other experiments. D.S.B., S.G., and J.A.M. wrote the manuscript with input from others. ACKNOWLEDGMENTS All authors are full-time employees in Johnson & Johnson. Received: September 17, 2015 Revised: December 15, 2015 Accepted: January 4, 2016 Published: February 11, 2016 REFERENCES Colombo, S.F., Mazzo, F., Pistillo, F., and Gotti, C. (2013). Biogenesis, trafficking and up-regulation of nicotinic ACh receptors. Biochem. Pharmacol. 86, 1063–1073. Consortium, G.T.; GTEx Consortium (2015). Human genomics. The GenotypeTissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science 348, 648–660. Cooper, S.T., and Millar, N.S. (1997). Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit. J. Neurochem. 68, 2140–2151. Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990). A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 5, 847–856. Dineley, K.T., Pandya, A.A., and Yakel, J.L. (2015). Nicotinic ACh receptors as therapeutic targets in CNS disorders. Trends Pharmacol. Sci. 36, 96–108. Freedman, R. (2014). a7-nicotinic acetylcholine receptor agonists for cognitive enhancement in schizophrenia. Annu. Rev. Med. 65, 245–261. Gotti, C., and Clementi, F. (2004). Neuronal nicotinic receptors: from structure to pathology. Prog. Neurobiol. 74, 363–396. Green, W.N., and Millar, N.S. (1995). Ion-channel assembly. Trends Neurosci. 18, 280–287.
Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 7
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
Halevi, S., Yassin, L., Eshel, M., Sala, F., Sala, S., Criado, M., and Treinin, M. (2003). Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression. J. Biol. Chem. 278, 34411–34417. Hogg, R.C., Raggenbass, M., and Bertrand, D. (2003). Nicotinic acetylcholine receptors: from structure to brain function. Rev. Physiol. Biochem. Pharmacol. 147, 1–46. Hurst, R.S., Hajo´s, M., Raggenbass, M., Wall, T.M., Higdon, N.R., Lawson, J.A., Rutherford-Root, K.L., Berkenpas, M.B., Hoffmann, W.E., Piotrowski, D.W., et al. (2005). A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J. Neurosci. 25, 4396–4405. Hurst, R., Rollema, H., and Bertrand, D. (2013). Nicotinic acetylcholine receptors: from basic science to therapeutics. Pharmacol. Ther. 137, 22–54. Jackson, A.C., and Nicoll, R.A. (2011). The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70, 178–199. Jackson, L.P., Lewis, M., Kent, H.M., Edeling, M.A., Evans, P.R., Duden, R., and Owen, D.J. (2012). Molecular basis for recognition of dilysine trafficking motifs by COPI. Dev. Cell 23, 1255–1262. Kassner, P.D., and Berg, D.K. (1997). Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells. J. Neurobiol. 33, 968–982. Koperniak, T.M., Garg, B.K., Boltax, J., and Loring, R.H. (2013). Cell-specific effects on surface a7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein. J. Neurochem. 124, 300–309. Kuryatov, A., Olale, F., Cooper, J., Choi, C., and Lindstrom, J. (2000). Human a6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39, 2570–2590.
Millar, N.S. (2008). RIC-3: a nicotinic acetylcholine receptor chaperone. Br. J. Pharmacol. 153 (Suppl 1 ), S177–S183. Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M., Salas, R., Molles, B., Marubio, L., Roth, U., et al. (2007). Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J. Neurochem. 102, 479–492. Nguyen, M., Alfonso, A., Johnson, C.D., and Rand, J.B. (1995). Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase. Genetics 140, 527–535. Orr-Urtreger, A., Go¨ldner, F.M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J.A., Patrick, J.W., and Beaudet, A.L. (1997). Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J. Neurosci. 17, 9165– 9171. Picciotto, M.R. (2003). Nicotine as a modulator of behavior: beyond the inverted U. Trends Pharmacol. Sci. 24, 493–499. Role, L.W., and Berg, D.K. (1996). Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16, 1077–1085. Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M., and Lindstrom, J. (1990). Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron 5, 35–48. Schuller, H.M. (2009). Is cancer triggered by altered signalling of nicotinic acetylcholine receptors? Nat. Rev. Cancer 9, 195–205. Schwartz, R.D., and Kellar, K.J. (1983). Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science 220, 214–216. Tomita, S., and Castillo, P.E. (2012). Neto1 and Neto2: auxiliary subunits that determine key properties of native kainate receptors. J. Physiol. 590, 2217– 2223.
Kuryatov, A., Mukherjee, J., and Lindstrom, J. (2013). Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional a7 AChRs. PLoS ONE 8, e62246.
Tran, P.V., Georgieff, M.K., and Engeland, W.C. (2010). Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa. Endocrinology 151, 4852–4860.
Le Nove`re, N., Corringer, P.J., and Changeux, J.P. (2002). The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J. Neurobiol. 53, 447–456.
Wang, H., Yu, M., Ochani, M., Amella, C.A., Tanovic, M., Susarla, S., Li, J.H., Wang, H., Yang, H., Ulloa, L., et al. (2003). Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421, 384–388.
Lester, H.A., Xiao, C., Srinivasan, R., Son, C.D., Miwa, J., Pantoja, R., Banghart, M.R., Dougherty, D.A., Goate, A.M., and Wang, J.C. (2009). Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery. AAPS J. 11, 167–177.
Williams, M.E., Burton, B., Urrutia, A., Shcherbatko, A., Chavez-Noriega, L.E., Cohen, C.J., and Aiyar, J. (2005). Ric-3 promotes functional expression of the nicotinic acetylcholine receptor a7 subunit in mammalian cells. J. Biol. Chem. 280, 1257–1263.
Lindstrom, J. (1997). Nicotinic acetylcholine receptors in health and disease. Mol. Neurobiol. 15, 193–222.
8 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
Zheng, Y., Mellem, J.E., Brockie, P.J., Madsen, D.M., and Maricq, A.V. (2004). SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans. Nature 427, 451–457.
Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO Highlights d
d
d
d
Genomic screening identifies NACHO as a mediator of a7 nAChR functional expression NACHO promotes folding and surface expression of a7 nAChRs
Authors Shenyan Gu, Jose A. Matta, Brian Lord, Anthony W. Harrington, Steven W. Sutton, Weston B. Davini, David S. Bredt
NACHO is a neuronal ER-resident protein that enhances a7 nAChR biogenesis
Correspondence
Alpha7 nAChR assembly and function in brain require NACHO
In Brief
[email protected]
Gu et al. used high-throughput screening to identify NACHO, a unique mediator of a7 nACh receptor assembly, biogenesis, and membrane insertion. Complete absence of functional a7 receptors in NACHO knockout mice establishes the essential role for this novel mechanism.
Gu et al., 2016, Neuron 89, 1–8 March 2, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2016.01.018
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
Neuron
Report Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO Shenyan Gu,1,2 Jose A. Matta,1,2 Brian Lord,1 Anthony W. Harrington,1 Steven W. Sutton,1 Weston B. Davini,1 and David S. Bredt1,* 1Neuroscience
Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2016.01.018 2Co-first
SUMMARY
Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing a7-type nAChR signaling improves symptoms in Alzheimer’s disease and schizophrenia. The pharmaceutical study of a7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie a7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of a7 receptors. NACHO promotes a7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts a7 receptor assembly and abolishes a7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors. INTRODUCTION Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate diverse physiological responses including pain processing and cognitive functions (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996). The a7 subtype is especially important, as this receptor not only participates in learning and memory (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996), but also underlies a cholinergic anti-inflammatory pathway relevant to autoimmune disorders (Wang et al., 2003) and stimulates cell proliferation and angiogenesis in lung and pancreatic cancers (Schuller, 2009). Upregulation of nAChRs represents a therapeutic goal and likely contributes to the pharmacology (Lester et al., 2009; Schwartz and Kellar, 1983) of cholinergic medicines in Alzheimer’s disease and schizophrenia (Dineley
et al., 2015; Hurst et al., 2013). Achieving this goal requires elucidation of fundamental mechanisms that control nAChR biogenesis. Nicotinic AChRs are members of the Cys-loop receptor family that include glycine, GABA-A, and 5-HT3 receptors, which all contain five subunits arranged in a ring surrounding the ion channel pore (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997). Assembly of nAChRs is a slow and tightly regulated process (Green and Millar, 1995) in which subunits first undergo a series of post-translational modifications followed by subunit-subunit interactions to form functional pentamers. Several general protein chaperones including BiP, ERp57, and calnexin modulate the trafficking and maturation of nAChRs (reviewed in Colombo et al., 2013). These ERresident proteins modulate biogenesis of numerous transmembrane proteins, and their precise roles in assembly of nAChR remain uncertain. Alpha7 nACh receptors are calcium-permeable homo-pentamers (Gotti and Clementi, 2004; Hogg et al., 2003; Le Nove`re et al., 2002; Lindstrom, 1997; Picciotto, 2003; Role and Berg, 1996), and they do not properly oligomerize or functionally express in almost all cell lines (Cooper and Millar, 1997; Kassner and Berg, 1997; Koperniak et al., 2013; Kuryatov et al., 2013). In C. elegans, resistance to inhibitors of cholinesterase-3 (Ric3) is required for nAChR activity (Nguyen et al., 1995). Whereas the mammalian Ric-3 homolog modestly increases a7 activity and has diverse effects on other co-expressed nACh and 5-HT3 receptors (Halevi et al., 2003; Millar, 2008), it is neither necessary (Koperniak et al., 2013) nor sufficient (Kuryatov et al., 2013) for efficient assembly of mammalian a7 receptors. Although some neurotransmitter receptors have auxiliary subunits that control receptor trafficking and channel gating (Jackson and Nicoll, 2011; Tomita and Castillo, 2012), no essential assembly proteins have previously been identified for a mammalian neurotransmitter receptor. RESULTS High-Throughput Screening Identifies NACHO as a Mediator of a7 Functional Expression To identify novel regulators of nAChR assembly and function, we utilized human embryonic kidney 293T (HEK) cells for highthroughput screening. In HEK cells transfected with a7 alone, ACh-evoked calcium influx, quantified with a fluorescence imaging plate reader (FLIPR), was undetectable, and application of Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 1
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
ACh + the a7 potentiator PNU-120596 (Hurst et al., 2005) produced a small calcium signal (Figure S1A). For FLIPR screening in a 384-well plate format, ACh (25 mM) and ACh plus PNU-120596 (10 mM) were sequentially applied to HEK cells in wells that had been individually co-transfected with a7 plus one of 3,880 clones from a cDNA library encoding almost all human transmembrane and secreted proteins (Origene). The single library clone that most profoundly augmented ACh + PNU-120596-induced responses (Figure S1A) derived from TMEM35, also termed unknown factor-1 (TUF-1), which encodes a small (167 amino acid) uncharacterized four-pass transmembrane protein (Figures S2A and S2B), whose expression in adrenal zona glomerulosa cells is increased by sodium depletion (Tran et al., 2010). We named this novel nAChR regulator (NACHO). To evaluate the effect of NACHO on a7 receptors, we performed electrophysiological experiments. Consistent with published studies (Cooper and Millar, 1997; Williams et al., 2005), we could not detect ACh-evoked currents in HEK cells transfected with a7 alone. Strikingly, co-transfection of NACHO with a7 yielded large, rapidly desensitizing ACh-evoked currents (Figures 1A and 1B). NACHO also significantly increased ACh-evoked currents in HEK cells co-transfected with a4 + b2 nAChR subunits (Figures 1A and 1B), which form hetero-pentameric receptors (Gotti and Clementi, 2004). In contrast, NACHO had no significant effect on glutamate- or 2-methyl-5-HT-evoked currents in cells co-transfected with NACHO and GluA1 or 5-HT3A/B receptors, respectively (Figures 1A and 1B). Furthermore, NACHO did not affect other ionotropic receptors tested including NMDA receptors, kainate receptors, TrpV1, and TrpM8 (Figure S1B). We asked whether NACHO increases surface expression of nAChR proteins. Therefore, we engineered in extracellular HA-tags that do not interfere with channel function and enable immunofluorescent detection of surface receptors. In HEK cells transfected with such a HA-tagged a7 construct alone, no labeling was detected following incubation of unpermeabilized cells with an anti-HA antibody (Figures 1C and 1D). Remarkably, cotransfection of NACHO yielded strong surface labeling for a7 (Figures 1C and 1D). We next evaluated the effect of NACHO on surface expression of HA-tagged a4b2, GluA1, and 5-HT3A/ B proteins. Consistent with our electrophysiological data, NACHO markedly increased a4b2 surface protein levels and had no effect on GluA1 or 5-HT3A/B (Figures 1C and 1D). NACHO is distantly related to only one human gene, ZMYM6NB, which also encodes a small, multi-pass transmembrane protein. We found that ZMYM6NB does not enhance the functional expression of a7 in co-transfected HEK cells (Figures S3A and S3B). A single potential ortholog of NACHO is present in the genomes of all vertebrates and some lower organisms. The NACHO homolog in Drosophila melanogaster (DNACHO) shares just 37% identity with human NACHO, yet it fully augments both functional and surface expression of co-transfected human a7 (Figures 1B and 1D). NACHO Promotes Assembly of a7 nAChR and Works Synergistically with Ric-3 We compared functional effects of NACHO with Ric-3, which is essential for nAChR responses in C. elegans (Nguyen et al., 1995) but whose mammalian homolog has mixed effects on a7, 2 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
a4b2, and 5-HT3 receptors (Halevi et al., 2003; Millar, 2008). In transfected cell lines, mammalian Ric-3 only modestly enhances a7 activity (Kuryatov et al., 2013). Endogenous Ric-3 is absent from hippocampal dentate gyrus neurons that are enriched in a7 (Halevi et al., 2003). Recent studies showed that complete knockdown of Ric-3 does not diminish a7 function in rat pituitary cells (Koperniak et al., 2013) demonstrating that endogenous Ric-3 is not essential. In whole-cell recordings from a7 co-transfected HEK cells, NACHO induces much larger ACh-evoked currents than does Ric-3 (Figures 2A and 2B). Interestingly, when both NACHO and Ric-3 are co-transfected, they synergistically increase ACh-evoked currents from a7 receptors, suggesting that they work through distinct mechanisms (Figures 2A and 2B). Assembly of a7 receptors can be quantified with a-bungarotoxin (a-Bgt), a snake venom component, which only binds to properly-folded a7 receptors (Couturier et al., 1990; Schoepfer et al., 1990). We used a-Bgt and a7 surface labeling, to compare effects of NACHO and Ric-3 on folding and trafficking of cotransfected a7-HA. In HEK cells transfected with a7-HA alone, neither surface a7 protein (purple) nor fluorescent a-Bgt binding before (red) or after (green) cell permeabilization could be detected (Figure 2C). In cells co-transfected with Ric-3 and a7-HA, a-Bgt binding and surface a7 labeling are also below our detection limits (Figure 2C). In striking contrast, co-transfection of a7-HA with NACHO yields abundant a-Bgt binding and a7 protein expression on the surface of unpermeabilized cells (Figure 2C). Following permeabilization of these a7 + NACHO co-transfectants, additional a-Bgt binding occurs (Figure 2C), which indicates the presence of folded intracellular a7 receptors that are not apparent without permeabilization (Figure 2C0 ). Cotransfection of NACHO and Ric-3 together with a7-HA further increases both surface a-Bgt binding and surface a7 protein levels and has no further effect on intracellular a-Bgt binding sites (Figure 2C). Immunofluorescent labeling for a7 following permeabilization shows that total a7 levels are similar in all transfection conditions (Figure 2C, cyan). NACHO Is a Neuronal ER-Resident Protein that Enhances a7 Biogenesis NACHO mRNA is uniquely enriched in brain (GTEx Project [Consortium, 2015]) and is discretely expressed in specific regions including hippocampus, cerebral cortex, and olfactory bulb (Figure S4A). Immunoblotting of rat hippocampal membranes or NACHO-transfected HEK cells with a NACHO antibody detects a protein band migrating at 18 kDa (Figure S4B), consistent with the predicted size of NACHO protein. This band is not present in rat liver or untransfected HEK cells. Importantly, this band is present (Figure S4B) in two neuroendocrine cell lines (PC12 and GH4C1) that can functionally express transfected a7 (Cooper and Millar, 1997; Koperniak et al., 2013). Immunofluorescent labeling of cultured hippocampal cells shows that NACHO occurs in neurons (identified with MAP2) and is not present in astrocytes (identified with GFAP) (Figure 3A). Higher-power magnification shows that NACHO co-localizes with an ER-resident protein disulfide isomerase (PDI) (Figure 3B). This ER localization of NACHO may be mediated through its extreme C-terminal tail (Figure S2A), which contains a canonical recognition sequence (KxKxx) that typically binds to b’-COP
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
B
C
D
Figure 1. NACHO Promotes Surface Expression of Functional nAChRs (A) Representative whole-cell recordings from HEK cells co-transfected with GFP and cDNAs encoding a7 /+NACHO; a4b2 /+NACHO; GluA1 /+NACHO; or 5-HT3A/B /+NACHO. (B) Summary graphs of agonist-evoked peak currents in HEK cells transfected as indicated (mean ± SEM). p values of comparisons ± NACHO: a7 p = 0.015, a4b2 p = 0.002, GluA1 p = 0.60, 5-HT3A/B p = 0.23. DNACHO represents the Drosophila melanogaster homolog (p = 0.028 ± DNACHO). (C) HEK cells were transfected as in (A) but with extracellular, HA-tagged receptors and were fluorescently labeled without permeabilization with anti-HA. (D) Quantification of surface labeling normalized to GFP (mean ± SEM, n = 5), p < 0.0001 ± NACHO in a7 or a4b2.
subunits for Golgi-to-ER retrieval (Jackson et al., 2012). Surface biotinylation on cultured hippocampal neurons did not detect NACHO protein on the surface of neurons (Figure 3C), consistent with NACHO being an intracellular (ER) protein. In a7-transfected HEK cells with or without Ric-3, all a7 protein is sensitive to Endo H glycosidase (Figure 3D), indicating that a7 in these cells does not progress properly from ER through Golgi. By contrast, co-transfection with NACHO generates an Endo
H-resistant population of a7 (Figure 3D), implying that NACHO enables a7 maturation. Ric-3 further enhances the effect of NACHO on a7 maturation (Figure 3D). Whereas NACHO promotes a7 assembly and biogenesis, we found that NACHO does not alter the desensitization or deactivation kinetics of either a7 (Figures S5A and S5B) or a4b2 (Figures S5D and S5E). We also investigated recovery from desensitization, which evaluates response to ACh at increasing intervals following a Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 3
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
C
B
C’
Figure 2. NACHO Mediates Assembly of a7 nAChR and Synergizes with Ric-3 (A) Whole-cell recordings from HEK cells transfected with cDNAs as indicated. (B) Quantification of ACh-evoked peak currents (mean ± SEM). Student’s t test p values: NACHO versus Ric-3 = 0.002; NACHO + Ric-3 versus NACHO = 0.0034. (C) Fluorescent labeling of HEK cells transfected as indicated. Surface staining of non-permeabilized cells used anti-HA (purple) and Bgt-555 (red). Following permeabilization, cells were labeled with Bgt-488 (green) to identify folded intracellular a7 and finally with anti-a7 (cyan) to quantify total receptor expression. (C0 ) As control, cells transfected with a7 + NACHO + Ric-3 were processed without permeabilization, which confirmed lack of labeling for Bgt-488 (green) after Bgt-555 (red).
desensitizing pulse of ACh. Again, NACHO does not affect recovery from desensitization for either a7 (Figure S5C) or a4b2 (Figure S5F). Furthermore, we performed immunoprecipitation experiments to assess whether NACHO and a7 tightly interact in hippocampal tissue. Because no available antibodies are suitable for western blotting of a7 in rodent brain (Moser et al., 2007), we detected a7 by [125I]a-Bgt binding. These experiments showed that NACHO immunoprecipitation does not bring down [125I]a-Bgt binding sites (Figure 3E). Conversely, a7 immunoprecipitation does not bring down NACHO protein. These results show that NACHO and a7 do not form a stable complex. Taken together, these data indicate that NACHO is not a surface-expressed a7 auxiliary subunit but rather is a neuronal, ER-localized chaperone for a7. Alpha7 Receptor Function in Brain Requires NACHO To evaluate functions for neuronal NACHO, we first identified an shRNA construct that effectively suppresses NACHO expres4 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
sion in hippocampal neurons (Figure 4B, purple). We quantified a7-mediated sustained currents in hippocampal neurons by applying ACh (1 mM) in the presence of PNU-120596 (1 mM), an a7 potentiator, that blocks the receptor desensitization. Remarkably, our shRNA-mediated knockdown of NACHO abolished the a7-mediated currents (Figure 4A). For a4b2, we quantified currents evoked by ACh (1 mM) that are sensitive to 10 mM dihydro-b-erythroidine (DhbE). We found that NACHO knockdown significantly decreases but does not eliminate these a4b2-mediated currents (Figure S6D), which fits our data in HEK cells that NACHO augments a4b2 currents (Figure 1B). These effects were specific as NACHO knockdown did not affect AMPA receptor-mediated currents (Figure 4A). Transfection of hippocampal neurons with an HA-tagged a7 yields abundant cell surface a7 and surface a-Bgt binding sites (Figure 4B, red). By contrast, HA-tagged a7 in neurons lacking NACHO are not present on the cell surface and do not form a-Bgt binding sites (Figure 4B).
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
B
C
E
D
Figure 3. NACHO Is an Intracellular Neuronal Protein that Promotes a7 Biogenesis (A) Immunofluorescent labeling of cultured rat hippocampal neurons shows that NACHO (red) occurs in MAP2-containing neurons (green) and does not colocalize with the astrocyte marker GFAP (purple). (B) Higher-power immunofluorescent labeling of hippocampal neurons shows co-localization of NACHO with the ER marker PDI (green). (C) Surface biotinylation of hippocampal neurons followed by immunoblotting shows that NACHO is exclusively intracellular. GluA1 and calnexin serve as references for cell surface and cytoplasmic proteins, respectively. Surface proteins were concentrated 5-fold (mean ± SEM, n = 3). (D) Glycosidase digestion of membranes from transfected HEK cells shows that NACHO promotes complex glycosylation of a7 as evidenced by resistance to Endo H treatment. Alone, Ric-3 has no effect on a7 glycosylation, but Ric-3 synergizes with NACHO. (E) Immunoprecipitation experiments show that anti-NACHO antibody does not pull down assembled a7, as detected by [125I]a-Bgt (mean ± SEM, n = 3), and that anti-a7 antibody does not pull down NACHO protein, as detected by immunoblotting.
We derived NACHO knockout mice (TMEM35tm1(KOMP)Vlcg) from the trans-NIH knockout mouse project (KOMP) repository (UC Davis). These knockout mice are viable, and they entirely lack NACHO protein and mRNA in brain (Figures S6A and S6B), whereas a7 mRNA is unaffected (Figure S6B). Electrophysiological recordings from acutely isolated hippocampal neurons showed complete absence of a7-mediated currents in NACHO knockouts. This deficit is specific, as AMPA receptors remain intact (Figure 4C). Furthermore, [125I]a-Bgt autoradiography shows that properly folded a7 receptors are undetectable in NACHO knockout brain (Figures 4D and S6C). DISCUSSION This study identifies NACHO as an essential factor for folding, maturation, and membrane insertion of a7. Our high-throughput screen of all predicted human transmembrane proteins indicates that NACHO has, by far, the greatest capacity to enhance assembly and function of a7. We find that the Drosophila NACHO ortholog effectively reconstitutes human a7 folding and function, which implies that the NACHO mechanism is
phylogenetically conserved. Most importantly, genetic deletion of NACHO abolishes functional a7 receptors throughout the brain. Whereas NACHO is required for a7 channel activity in brain, our heterologous cell and neuronal transfection experiments show that NACHO does not alter total a7 protein levels. The disposition of a7 protein in NACHO knockout mice is unknown because, unfortunately, no antibodies are suitable for immunostaining or immunoblotting of a7 in mouse brain (Moser et al., 2007). However, the complete loss of [125I]a-Bgt binding sites in NACHO knockout brain demonstrates absence of folded a7 pentamers. Cell biological properties distinguish NACHO from previously described neurotransmitter receptor partner proteins. Certain glutamate-gated ion channels, including AMPA and kainate receptors, also require neuronal accessory proteins (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). Best studied are transmembrane AMPA receptor regulatory proteins (TARPs), which, by analogy to NACHO, enhance surface trafficking of AMPA receptors (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). However, TARPs are not essential for AMPA receptor folding or tetramer assembly. Instead, TARPs are auxiliary Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 5
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
A
C
B
D
Figure 4. NACHO Is Required for Folding and Function of a7 Receptors in Brain (A) NACHO knockdown in rat hippocampal neurons selectively abolishes a7 channel activity. Whole-cell recordings of hippocampal neurons show that transfection with NACHO shRNA (red)—but not control shRNA (black)—eliminates ACh + PNU-120596 (PNU)-evoked currents (mean ± SEM, p < 0.0001). As control, NACHO knockdown does not affect currents evoked by Glu + cyclothiazide (CTZ) (mean ± SEM, p = 0.4562). (B) Fluorescent labeling shows that in a transfected neuron (green), NACHO shRNA effectively reduces endogenous NACHO protein (purple) and abolishes a-Bgt binding (red) and surface expression of co-transfected a7-HA (cyan). (C and D) NACHO knockout mice lack functional or folded a7 receptors. (C) Whole-cell recordings show that NACHO knockout (KO) mice (red) as compared to wild-type (black) do not display ACh + PNU-evoked currents but show normal responses to Glu + CTZ. Summary graphs for ACh + PNU-evoked (mean ± SEM, p < 0.0001) and Glu + CTZ-evoked currents in wild-type and NACHO knockout mice (mean ± SEM, p = 0.9817). (D) [125I]a-Bgt autoradiography on sagittal brain slices from wild-type and knockout mice shows that NACHO is essential for a7 receptor folding. CX, cortex; HP, hippocampus; SC, superior colliculus; IC, inferior colliculus; HY, hypothalamus.
subunits of AMPA receptors and associate with surface channels to control gating and pharmacology (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). By contrast, NACHO is fundamentally required for folding and assembly of a7 receptors and is the first essential, client-specific chaperone identified for a mammalian neurotransmitter receptor. The neuronal nAChR family comprises nine a and three b subunits, and all receptors other than a7-10 contain heteromeric subunits (Gotti and Clementi, 2004; Le Nove`re et al., 2002). Certain biologically important AChR subtypes, including a6b2containing receptors, cannot be functionally expressed in recombinant systems (Kuryatov et al., 2000). We also find that NACHO and Ric-3 are insufficient to promote functional expres6 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
sion of a6b2-containing nAChR (data not shown), which suggests that additional subtype-specific chaperones likely exist for these nAChR subtypes. Furthermore, selective chaperones may well mediate assembly of other neurotransmitter receptors. Neurotransmitter receptor auxiliary subunits and regulatory proteins have mostly been discovered by genetic screens (Nguyen et al., 1995; Zheng et al., 2004) and biochemical techniques (Jackson and Nicoll, 2011; Tomita and Castillo, 2012). Whereas these approaches have been applied to nAChRs, they did not identify NACHO. Our discovery of NACHO took advantage of a high-throughput screening strategy enabled by the recent availability of genome-wide cDNA libraries. Our new approach complements existing strategies and should help
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
identify assembly proteins and auxiliary subunits for numerous other receptors and ion channels. Genetic, biochemical, and pharmacological studies validate a7 as a therapeutic target for treating Alzheimer’s disease and schizophrenia (Dineley et al., 2015; Freedman, 2014; Gotti and Clementi, 2004). Challenges in reliably reconstituting functional a7 in cells lines have impeded pharmacological studies. Coexpression of NACHO with a7 solves this issue and will facilitate drug discovery. The chaperone function of NACHO may also represent a valuable drug target. Nicotine itself promotes maturation of several types of nAChRs (Schwartz and Kellar, 1983), and this likely contributes to nicotine’s inadvertent beneficial effects in certain cognitive and neurodegenerative conditions (Lester et al., 2009). Enhancing NACHO effects on nAChR assembly could provide a medical target devoid of nicotine’s addictive and toxic properties. EXPERIMENTAL PROCEDURES 2+
FLIPR Assay for Ca Influx in HEK Cells High-throughput FLIPR assays were performed using HEK cells in 384-well plates. Briefly, cells were seeded at 1 3 104 cells per well and transfected 4 hr after seeding with Fugene HD. After 2 days of incubation, cells were washed three times with assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES [pH7.4]) and loaded with Calcium5 dye (Molecular Device) and 1.25 mM probenecid for 1 hr at RT. After three washes, the plates were placed in the FLIPR stage. After establishing baseline, 25 mM acetylcholine was applied for a 1.5 min recording, then 25 mM acetylcholine plus 10 mM PNU120596 were applied for another 2.5 min recording. a-Bgt and Immunofluorescent Staining For surface staining, cells were incubated with 1 mg/ml a-Bgt or primary antibodies in culture medium for 30 min at 37 C. Cells were then washed and fixed with 4% paraformaldehyde. As indicated, cells were permeabilized with 0.3% Triton X-100 and incubated with antibodies or a-Bgt to probe intracellular components. Following incubation with secondary antibodies, cells were stained with DAPI for 5 min. Images were captured using a Zeiss M2 Imager or Zeiss LSM 700 confocal microscope. [125I] a-BTX Autoradiography [125I]a-BTX Autoradiography was performed as described (Orr-Urtreger et al., 1997). Non-specific binding was assessed by inclusion of 1 mM unlabeled a-BTX. At the end of the incubation, sections were washed four times and dried. Images were captured with b-Imager DFine (Biospacelab). Electrophysiology Forty-eight hours post-transfection, transfected cells were transferred to the recording chamber submerged in extracellular solution composed of 137 mM NaCl, 10 mM HEPES, 5 mM glucose, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2 (pH 7.4), and 300 mM mOsm. For cultured neurons, synaptic activity was blocked with TTX (0.5 mM), NBQX (10 mM; except when measuring whole-cell AMPA currents), and d-APV (50 mM). Whole-cell recordings used intracellular solution composed of 140 mM potassium gluconate, 10 mM HEPES, 4 mM Mg-ATP, 0.4 mM Na-GTP, and 0.6 mM EGTA (pH 7.3). Ultrafast acetylcholine was applied onto HEK cells using a piezo-driven perfusion system and theta glass (Siskiyou). Fast perfusion onto neurons used a Perfusion Fast-Step System (Warner Instruments). Acute hippocampal neurons were obtained from 7-week-old male mice. Horizontal slices, 300 mm thick, were obtained using a VT1200S microtome (Leica Biosystems). Slices were cut in ice-cold solution composed of 150 mM sucrose, 50 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 7 mM MgSO4, 2.5 mM KCl, 1.25 mM Na3PO4, and 0.5 mM CaCl2 equilibrated with 95% O2 and 5% CO2. Hippocampi were isolated and transferred to Hiberna-
teA (no calcium, BrainBits) solution containing 1 mg/ml papain (Worthington) and 0.5 mM Glutamax (Life Technologies) and digested at 37 C for 30 min. Slices were gently triturated with fire-polished Pasteur pipettes. The cell suspension was then plated onto coverslips and isolated neurons were used for whole-cell patch-clamp recordings. All dissociated cell recordings were done at room temperature and membrane potentials were held at 60 mV. Recordings used an AxoPatch 200B amplifier (Axon Instruments). For transfected HEK cells, signals were filtered at 10 kHz and digitized at 50 kHz. For neurons, signals were filtered at 2 kHz and digitized at 10 kHz. Data acquisition and online analysis were done using pClamp 9 (Axon Instruments). For recovery from desensitization, an initial desensitizing pulse of ACh was followed by a second pulse of ACh at varying time intervals. At each time interval, recovery from desensitization was expressed as the fraction of the current peak amplitude of the second pulse to the first pulse. Current decay kinetics and recovery from desensitization were fitted with double exponential functions and expressed as weighted decay time constants. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and six figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.neuron.2016.01.018. AUTHOR CONTRIBUTIONS D.S.B. supervised the project. J.A.M. and S.G. performed electrophysiological studies and data analyses. B.L. performed autoradiography. A.W.H. and S.G. performed immunofluorescent staining. W.B.D. and S.G. performed hippocampal neuron culturing and transfections. S.W.S. contributed to knockout animal derivation. S.G. performed all other experiments. D.S.B., S.G., and J.A.M. wrote the manuscript with input from others. ACKNOWLEDGMENTS All authors are full-time employees in Johnson & Johnson. Received: September 17, 2015 Revised: December 15, 2015 Accepted: January 4, 2016 Published: February 11, 2016 REFERENCES Colombo, S.F., Mazzo, F., Pistillo, F., and Gotti, C. (2013). Biogenesis, trafficking and up-regulation of nicotinic ACh receptors. Biochem. Pharmacol. 86, 1063–1073. Consortium, G.T.; GTEx Consortium (2015). Human genomics. The GenotypeTissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science 348, 648–660. Cooper, S.T., and Millar, N.S. (1997). Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit. J. Neurochem. 68, 2140–2151. Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990). A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 5, 847–856. Dineley, K.T., Pandya, A.A., and Yakel, J.L. (2015). Nicotinic ACh receptors as therapeutic targets in CNS disorders. Trends Pharmacol. Sci. 36, 96–108. Freedman, R. (2014). a7-nicotinic acetylcholine receptor agonists for cognitive enhancement in schizophrenia. Annu. Rev. Med. 65, 245–261. Gotti, C., and Clementi, F. (2004). Neuronal nicotinic receptors: from structure to pathology. Prog. Neurobiol. 74, 363–396. Green, W.N., and Millar, N.S. (1995). Ion-channel assembly. Trends Neurosci. 18, 280–287.
Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc. 7
Please cite this article in press as: Gu et al., Brain a7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO, Neuron (2016), http://dx.doi.org/ 10.1016/j.neuron.2016.01.018
Halevi, S., Yassin, L., Eshel, M., Sala, F., Sala, S., Criado, M., and Treinin, M. (2003). Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression. J. Biol. Chem. 278, 34411–34417. Hogg, R.C., Raggenbass, M., and Bertrand, D. (2003). Nicotinic acetylcholine receptors: from structure to brain function. Rev. Physiol. Biochem. Pharmacol. 147, 1–46. Hurst, R.S., Hajo´s, M., Raggenbass, M., Wall, T.M., Higdon, N.R., Lawson, J.A., Rutherford-Root, K.L., Berkenpas, M.B., Hoffmann, W.E., Piotrowski, D.W., et al. (2005). A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J. Neurosci. 25, 4396–4405. Hurst, R., Rollema, H., and Bertrand, D. (2013). Nicotinic acetylcholine receptors: from basic science to therapeutics. Pharmacol. Ther. 137, 22–54. Jackson, A.C., and Nicoll, R.A. (2011). The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70, 178–199. Jackson, L.P., Lewis, M., Kent, H.M., Edeling, M.A., Evans, P.R., Duden, R., and Owen, D.J. (2012). Molecular basis for recognition of dilysine trafficking motifs by COPI. Dev. Cell 23, 1255–1262. Kassner, P.D., and Berg, D.K. (1997). Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells. J. Neurobiol. 33, 968–982. Koperniak, T.M., Garg, B.K., Boltax, J., and Loring, R.H. (2013). Cell-specific effects on surface a7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein. J. Neurochem. 124, 300–309. Kuryatov, A., Olale, F., Cooper, J., Choi, C., and Lindstrom, J. (2000). Human a6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39, 2570–2590.
Millar, N.S. (2008). RIC-3: a nicotinic acetylcholine receptor chaperone. Br. J. Pharmacol. 153 (Suppl 1 ), S177–S183. Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M., Salas, R., Molles, B., Marubio, L., Roth, U., et al. (2007). Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J. Neurochem. 102, 479–492. Nguyen, M., Alfonso, A., Johnson, C.D., and Rand, J.B. (1995). Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase. Genetics 140, 527–535. Orr-Urtreger, A., Go¨ldner, F.M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J.A., Patrick, J.W., and Beaudet, A.L. (1997). Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J. Neurosci. 17, 9165– 9171. Picciotto, M.R. (2003). Nicotine as a modulator of behavior: beyond the inverted U. Trends Pharmacol. Sci. 24, 493–499. Role, L.W., and Berg, D.K. (1996). Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16, 1077–1085. Schoepfer, R., Conroy, W.G., Whiting, P., Gore, M., and Lindstrom, J. (1990). Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron 5, 35–48. Schuller, H.M. (2009). Is cancer triggered by altered signalling of nicotinic acetylcholine receptors? Nat. Rev. Cancer 9, 195–205. Schwartz, R.D., and Kellar, K.J. (1983). Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science 220, 214–216. Tomita, S., and Castillo, P.E. (2012). Neto1 and Neto2: auxiliary subunits that determine key properties of native kainate receptors. J. Physiol. 590, 2217– 2223.
Kuryatov, A., Mukherjee, J., and Lindstrom, J. (2013). Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional a7 AChRs. PLoS ONE 8, e62246.
Tran, P.V., Georgieff, M.K., and Engeland, W.C. (2010). Sodium depletion increases sympathetic neurite outgrowth and expression of a novel TMEM35 gene-derived protein (TUF1) in the rat adrenal zona glomerulosa. Endocrinology 151, 4852–4860.
Le Nove`re, N., Corringer, P.J., and Changeux, J.P. (2002). The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J. Neurobiol. 53, 447–456.
Wang, H., Yu, M., Ochani, M., Amella, C.A., Tanovic, M., Susarla, S., Li, J.H., Wang, H., Yang, H., Ulloa, L., et al. (2003). Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421, 384–388.
Lester, H.A., Xiao, C., Srinivasan, R., Son, C.D., Miwa, J., Pantoja, R., Banghart, M.R., Dougherty, D.A., Goate, A.M., and Wang, J.C. (2009). Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery. AAPS J. 11, 167–177.
Williams, M.E., Burton, B., Urrutia, A., Shcherbatko, A., Chavez-Noriega, L.E., Cohen, C.J., and Aiyar, J. (2005). Ric-3 promotes functional expression of the nicotinic acetylcholine receptor a7 subunit in mammalian cells. J. Biol. Chem. 280, 1257–1263.
Lindstrom, J. (1997). Nicotinic acetylcholine receptors in health and disease. Mol. Neurobiol. 15, 193–222.
8 Neuron 89, 1–8, March 2, 2016 ª2016 Elsevier Inc.
Zheng, Y., Mellem, J.E., Brockie, P.J., Madsen, D.M., and Maricq, A.V. (2004). SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans. Nature 427, 451–457.