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Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop ligand-gated ion channel superfamily. Muscle nAChR are heteropentamers that assemble from two α, and one each of β, γ, and δ subunits. Each subunit is composed of three domains, extracellular, transmembrane and intracellular. The transmembrane domain consists of four α-helical segments (M1–M4). Pioneering structural information was obtained using electronmicroscopy of Torpedo nAChR. The recently solved X-ray structure of the first eukaryotic Cys-loop receptor, a truncated (intracellular domain missing) glutamate-gated chloride channel α (GluClα) showed the same overall architecture. However, a significant difference with regard to the vertical alignment between the channel-lining segment M2 and segment M3 was observed. Here, we used functional studies utilizing disulfide trapping experiments in muscle nAChR to determine the spatial orientation between M2 and M3. Our results are in agreement with the vertical alignment as obtained when using the GluClα structure as a template to homology model muscle nAChR, however, they cannot be reconciled with the current Torpedo nAChR model. The vertical M2–M3 alignments as observed in X-ray structures of prokaryotic Gloeobacter violaceus ligand-gated ion channel and GluClα are in agreement. Our results further confirm that this alignment in Cys-loop receptors is conserved between prokaryotes and eukaryotes.
Cys-loop receptors are ligand-gated ion channels that conduct either anions or cations. The individual families are named after the ligand that gates them, γ-amino butyric acid receptors (GABAAR), nicotinic acetylcholine receptors (nAChR), serotonin receptors (5-HT3R), glycine receptors (GlyR). Many drugs in current clinical use target these receptors, and a variety of diseases involve Cys loop receptor dysfunction, for example epilepsy, anxiety, Alzheimer's and Parkinson's disease, schizophrenia, depression, attention deficit hyperactivity disorder, myasthenia gravis, psychosis, and nicotine addiction.
Pioneering detailed structural knowledge about these receptors was obtained by Nigel Unwin using electronmicroscopy of Torpedo nAChR, which show high sequence-identity with muscle nAChR (Miyazawa et al. 2003; Unwin 2005). The recent identification and subsequent X-ray crystal structure determination of prokaryotic homologues, the Gloeobacter violaceus ligand-gated ion channel (GLIC) and Erwinia chrysanthemi LGIC, have given high-resolution insights into the receptor structure (Tasneem et al. 2005; Hilf and Dutzler 2008, 2009). They have also depicted binding sites for numerous allosteric modulators (Hilf et al. 2010; Nury et al. 2011; Pan et al. 2012a,b). The usefulness of these structures for precise mechanistic insights is controversial, as the X-ray structures often do not reflect the functional state that is to be expected (Goyal et al. 2011; Parikh et al. 2011; Gonzalez-Gutierrez et al. 2012). Overall, the same core subunit architecture is found in the structures of metazoan and prokaryotic homologues: a conserved extracellular domain with two anti-parallel β-sheets and a transmembrane domain with four α-helical segments (Miyazawa et al. 2003; Unwin 2005; Hilf and Dutzler 2008, 2009; Bocquet et al. 2009; Hibbs and Gouaux 2011). Compared to eukaryotic Cys-loop receptors, the prokaryotic ones lack the eponymous disulfide-linked cysteines in the Cys-loop, as well as an intracellular domain (Tasneem et al. 2005). The intracellular domain in metazoans is contributed by a relatively long peptide (ca. 50–270 amino acids) between the transmembrane segments M3 and M4. The M3M4 loop in prokaryotes is barely longer than what is required to link the two transmembrane segments (3–14 amino acids). However, we have previously shown that the long intracellular domain in eukaryotic 5-HT3A and GABA-ρ1 receptors can be replaced by a heptapeptide (SQPARAA), the M3–M4 linker in GLIC inferred from multiple-sequence alignment studies, while retaining the ability of the receptors to fold, assemble, and function as ligand-gated ion channels upon expression in Xenopus laevis oocytes (Jansen et al. 2008). A comparable approach was utilized for the Caenorhabditis elegans glutamate-gated chloride channel α (GluClα) to obtain a crystallizable construct, in that the intracellular domain was replaced by a tripeptide (Hibbs and Gouaux 2011). This first eukaryotic Cys-loop receptor structure of GluClα surprisingly showed a vertical alignment between the channel-lining transmembrane segment M2 and the transmembrane segment M3 that was distinct from the one observed in the Torpedo nAChR structure (Unwin 2005; Unwin and Fujiyoshi 2012).
Several previous studies have indirectly performed experiments that may be used to assess the question of the vertical alignment between M2 and M3 in nAChR. However, none of them discussed the vertical alignment and/or the discrepancies. The laboratory of Grosman investigated the Cα distances in muscle nAChR of residues along the M1, M2, and M3 segments to the pore's long axis with a single-channel proton-transfer technique and found that these transmembrane segments only rearrange minimally during gating (Cymes et al. 2005; Cymes and Grosman 2008). While the rates of proton transfer for pre-M2 residues indicated that M2 started closer to the N-terminus than predicted by the Torpedo model, the vertical alignment between M2 and M3 cannot be directly assessed by these measurements. Several recent high-resolution NMR spectroscopy studies investigated the nAChR transmembrane domain. One studied the structure of the sole transmembrane domain (M1 to M4 segments) of the nAChR β2-subunit in hexafluoroisopropanol and arrived at the conclusion that the resulting four helix bundle “more resembled the structure of the TM domain in GLIC than that in the Torpedo β1 subunit” (Bondarenko et al. 2010). It was observed that M2 started two residues closer to the N-terminus than in the Torpedo structure. Next, a water-soluble transmembrane domain derived from the nAChR α1-subunit and additionally designed to promote monomeric structure was studied by NMR (Cui et al. 2012). This study specifically discussed the discrepancies in lengths of the various transmembrane segments between the NMR study of isolated individual transmembrane domains and the Torpedo structure. The latest NMR-derived structure resolved the transmembrane domains of α4 and β2 nAChR subunits in lauryldimethylamine-oxide micelles with comparable results. While none of the NMR spectroscopy papers directly addresses the discrepancies in their NMR-derived structures with the Torpedo structure with regard to the vertical alignment between M2 and M3, our analysis of the distances between the residues we investigated in the NMR-structures more closely resembles the distances observed in GluCl over those in Torpedo (Table 1).
Table 1. Distances between engineered Cys based on the X-ray GluCl and NMR nAChR structures
|Torpedo 2BG9 (A)||20.4||17.5||17.5||16.8||13.3||13.6||14.2|
|nAChR α4 2LLY||14.3||10.6||12.5||15.1||13.0||11.0||14.5|
|nAChR β2 2LM2||13.3||10.8||13.8||14.8||11.4||11.3||14.9|
|nAChR α1 WSA 2LKG||12.5||8.9||10.6||13.5||12.6||11.0||14.5|
|nAChR α1 WSA 2LKH||12.0||8.3||9.4||12.6||11.8||10.5||13.8|
As the transmembrane domain is of great importance for studies involving a multitude of allosteric modulators like alcohols, general anesthetics, neurosteroids, and other synthetic and natural compounds, we sought to address experimentally the vertical alignment of M2 and M3 in nAChR with this study. Here, we use disulfide trapping in α-subunits from mouse muscle nAChR that have 77.3% sequence identity and 88.1% similarity with α-subunits from Torpedo nAChR (93.4% identity and 100.0% similarity, respectively, for the sequences covering M2 and M3), to probe the proximity between engineered Cys in these two transmembrane segments. From our study that includes 13 Cys-pairs we conclude that the vertical alignment/register of a homology model of muscle nAChR built on the crystallizable engineered GluClα construct is in agreement with our experimental data whereas the Torpedo nAChR structural models conflicts with our results.
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- Materials and methods
The aim of this study was to experimentally probe the vertical alignment between the transmembrane segments M2 and M3 in nAChR to address a discrepancy between the Torpedo nAChR model and the recently published GluClα structure in this area (Unwin 2005; Hibbs and Gouaux 2011). The vertical alignment/register between M2 and M3 is crucial, as the transmembrane segments M1–M4 come together to contribute to intra- and inter-subunit binding sites for allosteric ligands. The antiparasitic agent ivermectin binds to an inter-subunit crevice lined by M1, M2, and M3 in GluClα, and likely in a similar fashion to other Cys-loop receptors (Krause et al. 1998; Adelsberger et al. 2000; Shan et al. 2001; Hibbs and Gouaux 2011). General anesthetic and neurosteroid binding site(s) in GABAAR have been mapped to inter- and intra-subunit sites contributed by M1, M2, and M3, using mutagenesis, Cys-scanning techniques, and photolabeling (Belelli et al. 1997; Bali and Akabas 2004; Hosie et al. 2006; Li et al. 2006; Bali et al. 2009; Chiara et al. 2012) and in the prokaryotic GLIC the general anesthetic site was identified by X-ray crystallography to an intra-subunit site that is linked to an inter-subunit site by a so-called ‘linking channel’ (Nury et al. 2011). Similarly, other allosteric modulators like alcohols and PNU-120596 have been shown to interact with positions in the transmembrane domain (Mihic et al. 1997; Lobo et al. 2004; Young et al. 2008; daCosta et al. 2011). These studies investigating the location of binding site(s) for allosteric ligands to Cys-loop receptors that interact with the transmembrane domain exemplify the importance of determining the vertical alignment and orientation between individual transmembrane segments.
The Torpedo nAChR atomic model was obtained by electronmicroscopy of tubular crystals grown from the postsynaptic membranes of the electric organ of Torpedo marmorata (Miyazawa et al. 2003; Unwin 2005). It is generally expected that several factors that may be problematic with X-ray crystallographic structure determination, like genetic engineering, over-expression in a heterologous host, nonnative detergent solubilization, lipid reconstitution, and other artificial crystallization conditions are not relevant for the Torpedo structure. To obtain the GluCl α-subunit structure several experimental hurdles had to be overcome (Hibbs and Gouaux 2011). As all eukaryotic Cys-loop receptors GluCl α-subunits contain a long intracellular domain between M3 and M4 that only consists out of three to 14 amino acids in prokaryotic members. This 58 amino acid long intracellular domain in GluClα was replaced with an Ala-Gly-Thr tripeptide in the crystallized GluClα construct. In addition, 41 amino acids of the N-terminus and 6 of the C-terminus were removed. Besides this extensive genetic engineering, the over-expression in insect cells (Sf9), extraction from membranes with n-dodecyl-B-D-maltopyranoside (C12M), and artificial crystallization conditions - extensive contacts with the Fab fragments, addition of nonnative lipids as well as polyethylene glycol, the acidic pH of 4.5, and the temperature of 4°C – may distort physiologically relevant structures. In particular, it is not clear whether the drastic replacement of the intracellular domain with a tripeptide lead to changes in the length of M3 and/or M4 and also their orientation and spatial alignment. It has been shown previously that M4 of Torpedo nAChR can be replaced by a different unrelated α-helical peptide, a hydrophobic transmembrane segment from stomatitis virus glycoprotein or human interleukin-2 receptor, while retaining the ability of the whole receptor to function as a ligand-gated ion channel upon expression in X. laevis oocytes (Tobimatsu et al. 1987).
Disulfide trapping has been widely applied to investigate proximity and mobility of engineered Cys-pairs in both soluble and membrane proteins (Falke and Koshland 1987; Matsumura et al. 1989; Careaga and Falke 1992b; Pakula and Simon 1992; Zhan et al. 1994; Gruber and Capaldi 1996; Danielson et al. 1997; Krovetz et al. 1997; Yu et al. 1999; Elling et al. 2000; Horenstein et al. 2001, 2005; Shapovalov et al. 2003; Bera and Akabas 2005; Chen et al. 2006; Yang et al. 2007). Here, we use disulfide bond formation as a measure for proximity. The maximum Cβ–Cβ distance observed in protein disulfide bonds is 4.6 Å (Sowdhamini et al. 1989). Therefore, for a disulfide bond to form, the respective Cys β-carbons have to come within 4.6 Å of one another. Additional constraints arise from factors like side-chain bonding, local steric barriers, and orientational constraints resulting from the transition state (Careaga and Falke 1992a,b). Generally, the separation distance of the α-carbons is assessed, and expected to be < 5.6 Å for disulfides to form. In this study, we constructed pairs of engineered Cys residues with site-directed mutagenesis, with one Cys in the muscle nAChR α-subunit transmembrane segment M2 at position T254C, and 13 different positions in M3 of the same subunit. All α-subunit constructs expressed, folded, assembled, and trafficked to the plasma membrane and formed functional ACh-gated ion-channels after heterologous expression in X. laevis oocytes, as evidenced by outward currents recorded upon application of ACh in two-electrode voltage clamp experiments. For none of the Cys-pairs disulfide bond formation was observed in the absence of the redox catalyst Cu : Phen, as: (i) repetitive ACh-applications produced stable current responses, and (ii) the application of the reducing agent DTT to oocytes from which a stable ACh-response had been recorded did not produce a significant change in current amplitude for wild-type, single or double mutants. Here, we provide evidence for disulfide cross-linking between engineered Cys pairs, one at position T254C and the other in M3. The Cys pairs that could be cross-linked by application of the redox catalyst Cu : Phen, M2 T254C, and M3 M278C, as well as M2 T254C and M3 L279C, clearly favor the vertical alignment as observed in the GluClα structure. Additional evidence for cross-linking of Cys pairs is provided by the fact that the reducing agent DTT could reverse the effect of Cu : Phen on the ACh-induced current amplitudes. The chelator EGTA, on the contrary, could not reverse Cu : Phen effects, indicating that chelation of Cu2+ by one or more engineered Cys and/or other amino acids did not significantly contribute to the Cu : Phen effect on ACh-induced current amplitudes. Importantly, double Cys mutants that should have had their Cys in a favorable distance if the Torpedo structure were to be correct could not be cross-linked.
When the initial coordinates based on the electronmicrocopic studies of two-dimensional crystals of Torpedo nAChR were released (PDB# 1OED), an extended remark was included in the coordinates file ‘The link between M1 and M2 was poorly resolved and the trace here is almost certainly wrong in detail. … Users should bear in mind that because of the limited resolution the conformations of the side chains and their atomic coordinates are not individually reliable. Also the ends of the helices are uncertain by at least one residue’ (Miyazawa et al. 2003). The location that was described as the most problematic one, the M1 and M2 loop, is exactly where the discrepancy between the Torpedo atomic model and the GluCl structure originates. Hibbs and Gouaux noted ‘that in the α-subunit M2 pore-lining helix and the M3 α-helix, the nAChR amino acid assignment is off in register by 4 residues or ~1 turn of an α-helix beginning with the M1–M2 loop’ (Hibbs and Gouaux 2011). Importantly, a similar discrepancy between amino acid and structure-based alignment had been previously observed and described for the comparison between GLIC and nAChR (Corringer et al. 2010).
As stated earlier, the interface between the extracellular domain and the transmembrane domain is crucial for coupling ligand binding to gating. Coupling involves the M2–M3 linker, the β1–β2 loop, the β8–β9 loop, the Cys loop, and the pre-M1 linker (Lee and Sine 2005; Reeves et al. 2005; Mercado and Czajkowski 2006). In nAChR mutant cycle analysis has shown functional coupling between Glu45 and Val46, located in the β1–β2 linker, to Ser269 and Pro272, part of the M2–M3 loop (Lee and Sine 2005). Pro272 has also been implicated as a cis-trans isomerization switch for channel activation (Lummis et al. 2005). Based on the proximity of the residues in the 2BG9 cryo-EM structure, where Glu45 and Val46 straddle Pro272, it was inferred that the coupling was direct as opposed to allosteric. However, distant allosteric coupling has been observed in nAChR by mutant cycle analysis for positions separated by 50–60 Å (Gleitsman et al. 2008). This demonstrates that mutant cycle analysis does not necessarily require physical proximity of mutant cycle interacting residues. Functional coupling between residues that are far apart has been commonly seen (Alexiev et al. 2000). Our results imply a shift in the vertical alignment between M2 and M3 as compared to the 2BG9 Torpedo nAChR model. This will change the M2–M3 linker interactions in the GluClα-based nAChR homology model. An additional complication for the homology model is that sequence alignments suggest that the GluClα M2–M3 linker is one amino acid shorter than in the nAChR. In the mouse nAChR model based on GluClα, Glu45, and Val46 straddle Pro264. In this model Glu45 forms a salt bridge to Arg209 in the pre-M1 linker; this links together the inner and outer β-sheets of the extracellular domain (Lee and Sine 2005). These residues are conserved in the Cys-loop superfamily (Mercado and Czajkowski 2006) and are crucial for gating in both models.
In summary, our experimental data are in agreement with the vertical alignment between M2 and M3 as obtained in a homology model of muscle nAChR based on the recently published GluClα structure, but not as in the Torpedo nAChR structural model. As the vertical alignment between the prokaryotic GLIC and GluClα is in agreement, our current study implies the same alignment between all Cys-loop receptor homologues, both prokaryotic and eukaryotic. Overall, amino acids are shifted by four positions for the M2 and M3 segments, each, leading to the register between the two segments to be different by two α-helical turns when Torpedo nAChR and GluCl are compared. If the muscle nAChR is homology-modeled on the recently published GluClα structure this leads to all M2 residues moving up the α-helix by four residues towards the extracellular side and consequently all M3 residues moving down by four residues towards the intracellular side. Because of the low resolution of the 2BG9 Torpedo nAChR electron density, it is reasonable to model the relative orientation between M2 and M3 as described in this study. Our study is limited in that we focused on the vertical alignment between M2 and M3. However, our observations can be used to interpret previous studies and it provides a model on which to base additional investigations. Further studies utilizing the mutants with engineered Cys pairs that could be cross-linked in this study or other double Cys mutants may be useful in investigating relative motions of the M2 and M3 segments during channel gating as well as the points of contact between loops of the transmembrane and extracellular domain. At present, no available structure of Cys-loop receptors is perfect. They are either hampered by low resolution, prokaryotic origin or genetic engineering, indicating the clear need for atomic resolution eukaryotic structures.