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Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
Nicotinic acetylcholine receptors (nAChR)2 are ligand-gated ion channel neurotransmitter receptors. In mammals, they are expressed as pentameric combinations of homologous subunits, translated from 16 different genes (α1–α7, α9, α10, β1–β4, γ, δ, and ϵ). Functional diversity of nAChR is determined by subunit composition, producing nAChR subtypes with overlapping pharmacological and biophysical characteristics (1).
α4β2*-nAChR are the most prevalent central nervous system (CNS) nAChR subtype, comprising ≈70% of all rodent CNS nAChR (2) and are implicated in a wide range of normal and pathological functions, including learning, memory, mood, and nicotine dependence among others (3,–17). α4β2*-nAChR functionally interact with nicotine at concentrations found in smokers and are the target of varenicline, currently the most successful smoking cessation pharmacotherapy (12, 13). Initial studies suggested an (α4)2(β2)3 subunit stoichiometry for these nAChR (18, 19). However, more recent work indicates that both native and heterologously expressed α4β2-nAChR can exist in two isoforms with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, respectively, displaying high and (predominantly) low sensitivities (HS and LS) to activation by acetylcholine (ACh) (20,–25). The expression of α4β2-nAChR isoforms appears to be physiologically significant. For example, multiple epilepsy-associated α4 and β2 subunit mutants alter ratios of HS to LS α4β2-nAChR isoforms (17, 26), and agonists capable of preferentially stimulating LS α4β2-nAChR produce distinctive physiological effects (27,–29). Accordingly, a better understanding of the respective roles of HS and LS α4β2*-nAChR isoforms is likely to have considerable translational implications.
Agonist binding to nAChR is primarily driven by interactions with a set of six peptide loops located at subunit interfaces. Three (loops A–C) are contributed from the subunit on the principal or (+)-side of the interface, with the remaining three (loops D–F) being contributed from the subunit on the complementary or (−)-side of the interface. The (−)-side subunit is oriented counterclockwise from the (+)-side subunit when viewed from extracellular space (Fig. 1A) (20). A high degree of conservation of critical agonist binding residues is seen across subunits. This includes residues within interfaces that do not harbor conventionally recognized canonical α(+)/(−)β-type agonist binding pockets (30,–34). Both α4β2-nAChR isoforms host a pair of canonical orthosteric, high affinity α4(+)/(−)β2 agonist binding interfaces, but the LS (α4)3(β2)2 -nAChR isoform also contains a unique, non-canonical, α4(+)/(−)α4 agonist-binding site (34,–36). This α4(+)/(−)α4 site has lower affinity for ACh or nicotine than the α4(+)/(−)β2 site, making it responsible for the intrinsically biphasic ACh concentration-response profile of the LS (α4)3(β2)2-nAChR isoform. This complex CRC distinguishes it from the HS (α4)2(β2)3-nAChR isoform, which lacks an α4(+)/(−)α4 subunit interface and produces monophasic CRCs (34,–36).
Activation of LS α4β2-nAChR via interactions with a sufficiently high agonist concentration to engage all three binding sites confers large increases in per receptor function compared with the HS isoform α4β2-nAChR, which can only be activated by agonist binding at the common pair of α4(+)/(−)β2 sites (36). Importantly, activation of the HS phase of functional responsiveness by LS (α4)3(β2)2-nAChR can occur just by targeting the common pair of α4(+)/(−)β2 sites (either at agonist concentrations too low to engage the α4(+)/(−)α4 site or by highly selective agonists that do not engage the α4(+)/(−)α4 site at all). In this case, function per receptor closely resembles that of the HS (α4)2(β2)3-nAChR isoform (34, 36, 37). Although the α4(+)/(−)α4 interface retains the typical features of an nAChR agonist-binding site, it may therefore be thought of as effectively potentiating the response as would a co-agonist or positive allosteric modulator site (34,–36). These previous studies by ourselves and others have implicitly treated the two canonical α4(+)/(−)β2 agonist-binding sites as a functionally equivalent pair. However, as illustrated in Fig. 1A, the nAChR pentameric assemblies of each isoform are pseudosymmetrical; the subunits that contribute the α4(+)/(−)β2 agonist-binding sites are surrounded by different neighbors. Because of this, it seemed possible that the two canonical agonist-binding sites might contribute differentially to activation (both within each isoform and between the two isoforms).
To test this hypothesis, we used a concatemeric (linked subunit) nAChR approach. This allowed us to express pure populations of both HS and LS isoform α4β2*-nAChR and to systematically mutate agonist-binding residues of α4(+)/(−)β2 interfaces either individually or in combinations on either background (Fig. 1). Unlike our previous study (36), we wished to modify, not destroy, ligand binding at the targeted sites. Therefore, we systematically altered triplets of β2 subunit (−)-face E-loop residues to their non-conserved counterparts in the α4 subunit (β2(V136H,F144Q,L146T); β2HQT) (Fig. 1B). This can convert agonist binding at α4(+)/(−)β2 sites to closely resemble that at an α4(+)/(−)α4 agonist-binding site (34, 38). If our hypothesis is correct, then making equivalent E-loop modifications in nominally equivalent α4(+)/(−)β2 interfaces should produce different outcomes. We also reciprocally mutated α4 subunit (−)-face E-loop residues (α4(H142V,Q150F,T152L); α4VFL (34)) to examine the effects of this substitution inside, outside, or in the absence of an α4(+)/(−)α4 interface, on both HS and LS backbones (Fig. 1C).
We demonstrate that the contributions of α4(+)/(−)β2 agonist-binding sites differ substantially within the HS isoform and between the two different isoforms upon β2HQT substitution. We further demonstrate that, unexpectedly, the α4VFL mutation produces very similar functional changes whether it is inserted into the LS isoform-unique α4(+)/(−)α4 agonist-binding site or not. Evidence for other allosteric effects of subunit (−)-face, E-loop, alterations also is obtained. These new insights may provide further opportunities to develop compounds that pharmacologically distinguish between the isoforms and thus increase our understanding of each isoform's role in normal and disease physiology.
Sazetidine-A and A-85380 were kindly provided by Drs. Alan Kozikowski (University of Illinois, Chicago) and F. Ivy Carroll (Research Triangle Institute, Research Triangle Park, NC), respectively. All other reagents were purchased from Sigma, unless noted otherwise.
Unlinked nAChR human wild-type (WT) α4 (CHRNA4; NCBI reference sequence designation NM_000744.6) or β2 (CHRNB2; NM_000748.2) subunit cDNAs were transcriptionally optimized, synthesized, and sequenced by GeneArt (ThermoFisher Scientific, Inc., Waltham, MA). Each subunit was subsequently excised from the GeneArt shuttle vector with the restriction enzymes XbaI and NotI and subcloned into a modified pCI expression vector containing an SwaI linearization site (vector was a gift from Dr. Isabel Bermudez, Oxford Brookes University, Oxford, UK). The unlinked triple mutant α4(H142V,Q150F,T152L) (α4VFL) or β2(V136H,F144Q,L146T) (β2HQT) subunit cDNAs were also synthesized and sequenced by GeneArt as variants of their optimized WT parent subunits. These mutants were then subcloned into pCI as described previously.
Dr. Isabel Bermudez (Oxford Brookes University) provided a low sensitivity pentameric (LSP) α4β2-nAChR construct encoding concatenated human nAChR subunits in the order β2-α4-β2-α4-α4 (21). This construct, cloned into the previously mentioned modified pCI expression vector, served as the parent plasmid for each of the concatemers engineered for this study. This included the high sensitivity pentameric isoform (β2-α4-β2-α4-β2; HSP) α4β2-nAChR construct (Fig. 1). The basic construction strategy has been described in detail elsewhere (21, 36). Briefly, all but the first β2 subunit were absent their start codons and signal peptides, and all but the last were devoid of a stop codon. Each subunit was tethered to its neighbor by a short stretch of nucleotides encoding a series of 6 or 9 (Ala-Gly Ser)n repeats, engineered to ensure a total linker length (including the C-terminal tail of the preceding subunit) of 40 ± 2 amino acids. A unique set of six restriction sites either flanking the entire concatemer or approximately bisecting each linker between subunits was introduced along the pentamer sequence (Fig. 1A). This permitted replacement of individual subunits (or cassettes of multiple subunits if desired) using standard restriction digestion and ligation methods. GeneArt was used to design, synthesize, and sequence-verify optimized WT and mutant subunits for substitution into the parent construct. These new subunits were variants of the unlinked optimized subunits previously described, with the addition of the flanking AGS-repeat linkers and restriction sites required for correct placement within the resulting new pentameric concatemers. A unique restriction site was also introduced into each synthetic mutant sequence destined for ligation within a concatemer. This allowed for unambiguous verification of every new construct.
As described previously (36), both unlinked and concatenated plasmid cDNA constructs were SwaI-linearized, proteinase K-treated, column-purified, and transcribed into cRNA (mMessage mMachine T7 kit, Ambion, ThermoFisher Scientific, Waltham, MA). Each cRNA sample was DNase I-treated, column-cleaned, and gel-analyzed for size and quality. Stage V/VI Xenopus laevis oocytes, purchased from Ecocyte LLC (Austin, TX), were injected with biased ratio combinations of mRNAs encoding unlinked subunits (1 or 10 ng) or with 20 ng of mRNA encoding a concatemeric construct. Injected oocytes were incubated at 13 °C for 6–10 days before testing.
Methodology for obtaining CRCs and maximal current responses from nAChR-injected oocytes has been published elsewhere (36, 39, 40). Briefly, at least 6 days post-injection, oocytes were voltage-clamped at −70 mV with an Axoclamp 900A amplifier (Molecular Devices, Sunnyvale, CA). Recordings were sampled at 10 kHz (low pass Bessel filter, 40 Hz; high pass filter, direct current), and the traces were extracted and analyzed using Clampfit software (Molecular Devices). Any oocytes with leak currents >50 nA were discarded. Drugs were applied at a flow rate of 4 ml min−1 using a 16-channel, gravity-fed perfusion system with automated valve control (AutoMate Scientific, Inc., Berkeley, CA) in OR2 buffer (NaCl, 82.5 mm; KCl, 2.5 mm; MgCl2, 1 mm, HEPES 5 mm, pH 7.6) at 22 °C. Solutions were supplemented with atropine sulfate (1.5 μm) to block any muscarinic responses. Agonists were acutely perfused for 1 s with a 60-s washout between drug applications. In CRC analyses requiring a 5-min sazetidine-A (3.16 nm) pretreatment, both ACh and wash applications contained the same concentration of sazetidine-A. All concentration-response recording sessions included oocytes injected with non-mutant HSP and LSP concatemer controls (minimum of three oocytes per group) to account for day-to-day and batch-to-batch variability in functional expression levels.
Introduction of any mutation could potentially alter surface expression of the host nAChR. Any such changes would have corresponding effects on functional expression, independent of effects on function per receptor. Surface expression levels of each concatenated α4β2-nAChR isoform, and variant thereof, were quantified with 125I-mAb 295 in an oocyte binding assay. For consistency, the entire family of either LSP or HSP triple E-loop mutants was tested on the same day. LSP and HSP controls (containing no E-loop mutant subunits) were included in every experiment. mAb 295 is a monoclonal antibody that specifically recognizes correctly folded human, bovine, and rodent nAChR β2 subunits (41,–43). The protocol used has previously been described (26, 36, 44). In brief, following determination of expressed nAChR function by maximal stimulation (Imax, where Imax is peak current response) with ACh (EC100, 1 s), sets of six oocytes each expressing individual concatemeric α4β2-nAChR isoforms or variants thereof were sorted into a 24-well plate (one set per well). The accompanying OR2 buffer was aspirated from each well and replaced with 2 nm 125I-mAb 295 in OR2 (200 μl), supplemented with 10% heat-inactivated fetal bovine serum (to reduce nonspecific binding), and incubated with gentle agitation for 3 h at 22 °C. Washing was performed by aspiration of the radioactive solution and replacement with OR2 supplemented with 10% heat-inactivated fetal bovine serum (2 ml, ice-cold). The oocytes were then transferred to a fresh 24-well plate with the minimum possible volume of diluted radioactive solution. This wash protocol was repeated twice more. The thrice-washed oocytes were transferred to another fresh 24-well plate before being lysed overnight in 0.1% SDS, 0.01 n NaOH (0.5 ml), prior to scintillation counting at 85% efficiency using a Packard TriCarb 1900 Liquid Scintillation Analyzer (PerkinElmer Life Sciences). One or more wells of non-injected oocyte controls were included per assay plate to determine nonspecific binding. Nonspecific binding was subtracted from total binding determined in each of the other wells of the same plate, to calculate specific binding. Mean specific counts for sets of six HSP or LSP isoform-expressing oocytes were 885 and 469 cpm, respectively. This compared with mean nonspecific binding of 98 cpm per set of six uninjected oocytes. Specific cell-surface binding of 125I-mAb 295 was converted to nAChR surface expression per oocyte using the specific activity of the radioligand (initially 1150 Ci/mmol, but falling due to non-catastrophic decay of the radiolabel) and by accounting for two antibody-binding sites (two β2 subunits) per LSP isoform variant or three binding sites (three β2 subunits) per HSP variant (Fig. 1). For each experiment, the resulting binding data (femtomoles of nAChR/oocyte) were used to normalize Imax values (microamperes/oocyte; calculated as the mean of the individually measured Imax values obtained from the oocytes comprising the set of six used in each binding determination) to microampere/fmol. These specific function values were then compared among all E-loop mutant concatemers and to the non-E-loop mutant LSP and HSP controls.
Log10 EC50 values were determined from individual CRCs by non-linear least squares curve fitting (Prism 5.0; GraphPad Software, Inc., La Jolla, CA). Unconstrained monophasic or biphasic logistic equations were used to fit all parameters, including Hill slopes and fractional contributions of function attributable to HS or LS nAChR, where applicable. A sum-of-squares F-test was used to verify when data were better fit by the biphasic rather than monophasic model. Note that precision in determining ACh log10 EC50 values from biphasic CRCs (S.E. in log10 EC50 values between 0.1 and 0.35) is substantially lower than for monophasic CRCs (log10 EC50 S.E. values ≤0.07). One-way analysis of variance (ANOVA) and Bonferroni's multiple comparison tests were used to compare functional parameters across α4β2-nAChR variants (Prism 5.0 or SigmaPlot Version 12.5, Systat Software Inc., San Jose, CA).
As noted in the Introduction, the account describing how HS phase function arises in either LS or HS isoform α4β2-nAChR (34, 36) contains an implicit assumption. This is that the two α4(+)/(−)β2 agonist-binding sites found in both isoforms (see Fig. 1A) are functionally equivalent (both to each other in a given pentameric backbone, and between HS and LS α4β2-nAChR isoforms). Fully linked pentameric constructs provide the ability to substitute E-loop mutant subunits at any chosen position or combination of positions in HS or LS isoform α4β2-nAChR. Using these concatemeric constructs, we were therefore able to systematically test this assumption for the first time, by introducing the β2HQT E-loop mutant (located on the (−)-side of the β2 subunit) into defined positions within LSP and HSP α4β2-nAChR (Fig. 1B). The resulting family of constructs represents every possible permutation of the β2HQT triple mutant subunit cloned into the WT LSP and HSP parent constructs.
In the case of the LSP-based constructs, substitution of the (−)α4-like β2HQT mutant subunit into either of the α4(+)/(−)β2 agonist-binding sites (transforming the interface into something resembling the lower affinity α4(+)/(−)α4 interface) abolished HS component function (Fig. 2A). The same was true when β2HQT substitution was performed at both α4(+)/(−)β2 sites. The resulting monophasic CRCs produced by the three mutant LSP constructs had very similar log10 EC50 values to each other (EC50 ≈230 μm) and were approximately one-half log unit higher than the estimated log10 EC50 value for the LS component of LSP control (EC50 ≈60 μm; Fig. 2A, Table 1). In summary, β2HQT substitution effects on α4β2-nAChR LSP isoform function were both position- and copy number-independent; in every case the resulting function was purely LS component-like.
In striking contrast to the LSP series, the HSP-derived family of β2HQT-containing concatemers generated a complex set of CRCs that could be classified into three groups (Fig. 2B). The first group consisted of two concatemers hosting a single copy of β2HQT at positions 3 or 5, which yielded HS log10 EC50 values indistinguishable from that of the unmutated HSP control (EC50 ≈2 μm; Table 1). Note that the β2HQT subunit in position 3 is part of an α4(+)/(−)β2 binding pocket, although that in position 5 is not (Fig. 1B). In other words, these constructs had one (β2HQTp3) or no (β2HQTp5) α4/β2 interfaces converted to a mock α4(+)/(−)α4 interface, retaining one or both natural α4(+)/(−)β2 interfaces, respectively.
The second HSP mutant group had just one member, the HSP concatemer containing a single β2HQT subunit at position 1. This β2HQTp1 construct also had one α4(+)/(−)β2-to-mock α4(+)/(−)α4 interface conversion but, like the β2HQTp3 mutant, preserved one α4(+)/(−)β2 interface. This concatemer was the only HSP β2HQT mutant construct that generated a biphasic ACh CRC. The observed HS and LS phase EC50 values for this construct (≈1.8 and ≈60 μm, respectively) were indistinguishable from those of the LSP control (≈2.8 and ≈60 μm; Table 1). This biphasic response was so striking that it was further confirmed using two highly HS versus LS isoform-selective nicotinic agonists, A-85380 and the structurally related compound sazetidine-A. A-85380 demonstrates an unusually high degree of agonist selectivity between HS and LS isoform α4β2-nAChR (24, 36, 45). In response to acute A-85380 stimulation, the HSP β2HQT position 1 construct generated a clearly biphasic CRC with an ~1:1 distribution of HS and LS components (Fig. 3), confirming the initial observation of a biphasic ACh CRC. Sazetidine-A (46) activates only HS isoform α4β2-nAChR with high efficacy (47) and may be used to selectively occupy (36, 37) and inactivate (by desensitization) responses arising from α4(+)/(−)β2 interfaces (36). A desensitizing concentration of sazetidine-A (3.16 nm (36)) was used to pretreat oocytes expressing the HSP β2HQT position 1 concatemer prior to acute A-85380 exposure. This led to complete suppression of the HS portion of the response, confirming that the resulting CRC must arise from A-85380 activity at the remaining ((position 4) α4(+)/(−)β2 (position 3)) interface left within the position 1 HSP β2HQT construct.
The third group of HSP mutants consisted of all concatemers containing two copies of the β2HQT E-loop, plus that with all three positions mutated to the β2HQT (Fig. 2B and Table 1). All three doubly substituted constructs produced log10 EC50 values indistinguishable from each other or from that of the LS phase of unmutated LSP α4β2-nAChR (EC50 ≈60 μm). Interestingly, even a combination of β2HQT subunits placed in positions 3 and 5 (neither of which had any effect individually) resulted in loss of all HS phase function. The EC50 value for the triple β2HQT mutant HSP (≈190 μm) was like that of β2HQT mutants introduced into the LSP backbone (Fig. 2A and Table 1). Thus, in complete contrast to the effects of β2HQT mutant subunit incorporation on LSP α4β2-nAChR, effects in HSP α4β2-nAChR constructs were strongly influenced both by position and by copy number. Most importantly, effects on ACh CRC responses were radically different between the two constructs in which a single β2HQT mutant subunit was placed in either of the two nominally identical α4(+)/(−)β2 agonist binding pockets.
In addition to exploring effects of β2HQT substitution at defined subunit positions on agonist activation affinities, we also wanted to assess effects on amounts of nAChR function. We previously have shown that cell surface levels of HSP and LSP α4β2-nAChR isoforms in oocytes injected with equivalent levels of pentamer cRNA are comparable (36). This indicated that the observed differences in amount of HSP versus LSP function were almost exclusively due to differences in function per surface-expressed nAChR; i.e. due to the intrinsic properties of the α4β2-nAChR isoforms. We have also shown that introducing mutations at subunit interfaces can significantly affect surface expression of the resulting nAChR constructs (36). Accordingly, we normalized function to the amount of nAChR surface expression (measured using β2-specific monoclonal antibody binding).
For the LSP β2HQT mutant constructs, measurements of maximum ACh-induced function (Imax) alone showed significant losses of function for both the position 1-only and double mutant β2HQT mutant constructs, reducing Imax to a level resembling that of the unmutated HSP control construct (Fig. 4A). The LSP position 3-only β2HQT mutant construct, in contrast, produced an unchanged Imax value compared with the unmutated LSP construct. However, as shown in Fig. 4B, surface expression of all three LSP β2HQT mutant constructs was significantly suppressed by incorporation of the mutant subunit(s). When we compensated for this effect (Fig. 4C), it became apparent that function of both single mutant LSP β2HQT mutant constructs was significantly increased, to approximately double that of the unmutated LSP control, on a per nAChR basis (Table 1). Also, on a per nAChR basis, function of the position 1 + 3 double LSP β2HQT mutant construct was statistically similar to that of the unmutated LSP α4β2-nAChR construct (Fig. 4C and Table 1). As in the case of log10 EC50 values (Fig. 2A), β2HQT-mediated functional enhancement in LSP was position-independent. However, copy number did affect the per nAChR amount of function, unlike effects on agonist log10 EC50 values that were the same regardless of whether one or both positions contained β2HQT mutant subunits (Fig. 2A).
The family of HSP β2HQT concatemers again revealed a more complicated response to incorporation of mutant subunits (Fig. 5 and Table 1). The single copy β2HQT position 1 construct (the only α4β2 HSP mutant to produce a biphasic CRC; Fig. 2B) exhibited significantly lower surface expression and Imax. On a per nAChR basis, these effects combined to produce an apparent, but not-quite-significant, reduction in per nAChR function. In contrast, the position 3-only β2HQT mutant (which did not affect the ACh concentration-response outcome; Fig. 2B) significantly increased Imax compared with the unmutated HSP control, while having no effect on surface expression. On a per nAChR basis, this resulted in a statistically significant doubling of function. Although the non-agonist-binding interface position 5 β2HQT single mutant HSP construct showed a modest but marginally significant increase in Imax compared with the non-mutant HSP control, this did not quite retain significance on a per nAChR basis. Neither did this position 5-only mutant construct produce any effect on ACh concentration-response (Fig. 2B). Interestingly, per nAChR function of two of the HSP double-β2HQT mutant constructs (positions 1 + 3 and 3 + 5) did not significantly differ from that of HSP control. However, when the position 1 β2HQT subunit was included with the off-interface position 5 β2HQT subunit, in either the double and triple mutant configurations (e.g. positions 1 + 5, or positions 1 + 3+5), there was a highly significant, ≥60%, reduction in function per nAChR. To summarize, position- and copy-dependent β2HQT mutant subunit effects on per nAChR function again exposed asymmetries between the nominally similar α4(+)/(−)β2-binding sites within the HSP α4β2-nAChR isoform.
In addition to the just-explored α4(+)/(−)β2 agonist-binding sites common to HSP and LSP α4β2-nAChR, we also wished to specifically target the unique α4(+)/(−)α4 interface present between positions 4 and 5 within the LSP (Fig. 1A, left panel). A previous study (34) had addressed this question using unlinked α4VFL mutant subunits. However, the unlinked subunit approach altered all three α4 subunits in the α4β2-nAChR LS isoform. In contrast, use of concatemeric constructs allowed us to assess effects of α4VFL mutant incorporation at each available position, either singly or in combination (see Fig. 1C).
Unexpectedly, CRCs obtained from the family of α4β2-nAChR LSP α4VFL-containing constructs were uniformly monophasic (Fig. 6A). This was true whether single or multiple positions were substituted and was independent of which position(s) was altered. Even the three LSP variants where the α4(+)/(−)α4 interface E-loop was not mutated (α4VFL in positions 2, 5, or 2 + 5; see Fig. 1C) displayed the same outcome. Even more surprisingly, statistically indistinguishable EC50 values (between 7 and 12 μm) were obtained from all of the LSP α4VFL mutant constructs; these EC50 values were intermediate between those of the HS (0.6 μm) and LS (≈60 μm) phases of unmutated LSP function (Fig. 6A and Table 2). This was true even though there was no change in numbers of α4(+)/(−)β2 or α4(+)/(−)α4 interfaces or even when the α4(+)/(−)α4 interface was replaced by an additional mock α4(+)/(−)β2 interface. In summary, effects of the α4VFL E-loop mutant were completely independent of position and copy number in the LSP α4β2-nAChR isoform. A very similar outcome was observed when introducing α4VFL mutant subunits into the α4β2-nAChR HSP isoform (where none of the mutant subunits could be introduced into an agonist binding pocket; see Fig. 1C). In every case, CRCs of α4VFL HSP mutant constructs remained identical to that of the non-mutant HSP control (EC50 ≈2 μm; Fig. 6B and Table 2).
Although the agonist-pharmacology effects of α4VFL mutant subunit incorporation into LSP α4β2-nAChR were position- and copy number-independent, this was not the case for effects on function per receptor (Fig. 7). As shown in Fig. 7A, Imax values were significantly reduced for all α4VFL mutant LSP constructs. Qualitatively, the reduction of Imax values increased progressively from single- to double- to triple-substituted LSP constructs (Fig. 7A). Interestingly, reductions in Imax for each of the three single-substituted constructs were largely accounted for by loss of surface expression (Fig. 7B); no changes in per nAChR function compared with the unmutated LSP α4β2-nAChR control were measured for any of the single α4VFL-substituted constructs (Fig. 7C and Table 2). However, all three of the double α4VFL-substituted LSP constructs produced significantly less per nAChR function compared with the unaltered α4β2-nAChR LSP control. This loss of per nAChR function was primarily driven by an ~50% loss of Imax, because changes in surface expression were quite similar across the range of single, double, and triple α4VFL-substituted LSP constructs. As shown in Fig. 7C and Table 2, the loss of function/nAChR was the same in all three of the double α4VFL-substituted LSP constructs. The trend was extended in the case of the triple α4VFL-substituted LSP construct, where a further reduction (to ~3% of control) in per receptor function was seen (Fig. 7C and Table 2). In conclusion, function/nAChR effects of α4VFL substitution into α4β2-nAChR LSP are position-independent but are copy number-dependent.
This position-independent but copy number-dependent effect of α4VFL substitution was also seen in the α4β2-nAChR HSP background (Fig. 8). α4VFL substitution into either or both available α4 subunit positions suppressed Imax significantly compared with control and reduced nAChR surface expression by a little over 50% (Fig. 8, A and B). When function was normalized to a per nAChR basis, the two single-substituted α4VFL α4β2-nAChR HSP variants exhibited a similar not-quite-significant trend to lower function, although the double-substituted construct produced significantly less function (approximately a 75% reduction; Fig. 8C and Table 2) when compared with the unmodified α4β2-nAChR HSP construct.
As shown previously, α4β2-nAChR pentameric and concatemeric constructs can be expressed without degradation and/or rearrangement of subunit ordering (21, 35, 36). Importantly, these previous studies demonstrate that concatenated subunit pentameric constructs are capable of accurately replicating the salient functional pharmacological features of HS and LS α4β2-nAChR isoforms. To test this point further, we compared results from the concatemeric constructs used in this study to those obtained using unlinked α4 and β2 subunits. This was done using wild-type α4 and β2 subunits, and β2HQT and α4VFL mutant subunits, injected at ratios known to favor formation of HS or LS α4β2-nAChR isoforms. Further comparisons were made to previously published results using these mutant subunits (34). Where subunit compositions were comparable, the results remained extremely similar to each other across our concatemeric (Tables 1 and and2)2) and our unlinked nAChR subunit experiments (Table 3). Both levels of functional expression and ACh sensitivities match previous findings by others and for receptors of like construction whether from linked or unlinked subunits.
To determine whether the effects of E-loop mutations on ACh potency were unique, or whether they might be more generalizable, we performed an equivalent set of CRC experiments for nicotine (Fig. 9 and Table 4). Although nicotine is less discriminating between HS and LS phase function than ACh (24), very similar outcomes were clearly visible (compare outcomes in Fig. 9 to those in Figs. 2 and and6).6). Mutant constructs that exhibited increased ACh potency also showed increased nicotine potency. Mutant constructs that exhibited decreased ACh potency also did so for nicotine. Most compellingly, the position-1 β2HQT HSP backbone variant showed a significantly shallower Hill slope for nicotine activation and a significantly decreased log10 EC50 value. These are indicators of a composite CRC containing a new nicotine-evoked response component with lower nicotine sensitivity compared with the parent construct (as seen for the same construct when stimulated with the more HS versus LS phase-discriminating agonists ACh and A-85380 (Figs. 2 and and33).
In a pioneering study, it was first noted explicitly that LS isoform α4β2-nAChR produce a small proportion of intrinsic HS-like function (34). Our subsequent finding was that this fraction of HS phase function produced by the LS isoform was similar in terms of function per receptor to the entire response of HS isoform α4β2-nAChR (36). These data suggested that HS phase function in both α4β2-nAChR isoforms may be produced by activation of the pair of α4(+)/(−)β2 interfaces common to each isoform. This concept was reinforced by two further observations (36). First, HS phase function could be selectively eliminated by pre-desensitization with the strongly HS activation-preferring compounds sazetidine-A and A-85380. Second, mutations at the α4(+)/(−)α4 site of LS isoform α4β2-nAChR preferentially reduced LS phase function. However, both these studies, and a further one reinforcing the importance of α4(+)/(−)α4 site engagement for effective activation of LS isoform α4β2-nAChR (37) implicitly treated the two α4(+)/(−)β2 agonist-binding sites as an equivalent pair.
As outlined in the Introduction, our hypothesis stated that the two canonical agonist-binding sites might contribute differentially to activation (both within each isoform and between the two isoforms). This possibility arises because the environments in which the agonist sites are found are not identical; subunits that contribute the α4(+)/(−)β2 agonist-binding sites are surrounded by different neighbors. Please note that because our experiments with ACh and nicotine both produced very similar outcomes, we will henceforth predominantly discuss only our data obtained with ACh, for the sake of clarity. An overview of our major findings is presented in Fig. 10. It illustrates that insertion of the E-loop β2HQT mutant at one or the other of the two apparently equivalent α4(+)/(−)β2 agonist-binding sites in the HS α4β2-nAChR isoform produces different changes in ACh sensitivity and/or levels of function per surface nAChR. We also demonstrate that nominally identical changes made at the same α4(+)/(−)β2 interfaces in the LS α4β2-nAChR isoform give markedly different functional outcomes from those seen on the HS isoform background. We conclude that the two conserved α4(+)/(−)β2 agonist-binding sites contribute differently to HS α4β2-nAChR isoform function and between LS and HS α4β2-nAChR isoforms.
Introduction of β2HQT subunits has been proved to alter agonist binding at α4(+)/(−)β2 interfaces, making it resemble more closely binding at an α4(+)/(−)α4 site (34, 38). In this study, placement of β2HQT mutant subunits into the (−)-side of the conserved canonical α4(+)/(−)β2 agonist binding interfaces produced very similar outcomes in the LSP (α4)3(β2)2 isoform. For example, β2HQT insertion into either possible position produced indistinguishable changes in agonist pharmacology and per receptor function. These outcomes indicate that, in partial contradiction of our original hypothesis, the two canonical α4(+)/(−)β2 agonist-binding sites within the LS isoform α4β2-nAChR likely contribute in similar ways to receptor activation.
However, the same is not true in the context of the HS isoform. Here, β2HQT insertion into the first versus the second concatemeric α4(+)/(−)β2 agonist binding interface resulted in dramatically different ACh CRC outcomes. Effects on per receptor function also differed considerably between these two singly substituted HSP mutants. These results are the first demonstration that, despite the fact that the composition of these canonical α4(+)/(−)β2 agonist-binding sites is completely conserved, they make significantly different contributions to HS isoform α4β2-nAChR activation. This finding is in marked contrast to the position-independent situation in LS isoform α4β2-nAChR (Fig. 10). Together, these two outcomes confirm our original hypothesis.
The HSP-α4β2-nAChR construct contains no α4(+)/(−)α4 interfaces, so no direct effect would be anticipated on agonist binding following introduction of the E-loop α4VFL mutation into α4(−)-interfaces. As shown in Fig. 6B, this expectation was met; CRCs were indistinguishable across all of the α4β2-HSP constructs. Effects on surface expression and per receptor function were noted, and these were symmetrical (Figs. 7, B and C, and and10).10). Similar trends were observed for per receptor function.
However, results from the LSP-α4β2-nAChR construct family, all members of which do contain an α4(+)/(−)α4 interface, were much more surprising. Here, substitution of the α4VFL mutant subunit into any position, or combination of positions, resulted in an identical outcome (Fig. 6A). Similar to the outcome seen in the α4β2-HSP-nAChR context, LSP per receptor function was progressively reduced as the number of α4VFL mutant subunits introduced was increased, and no position dependence was seen (Figs. 7C and and10).10). These findings are surprising given that the α4(+)/(−)α4 interface within the α4β2-LS-nAChR isoform has been shown directly, by multiple approaches, to correspond to an agonist-binding site (35, 36). From these findings, we conclude that α4(−)-face E-loop contributions to α4β2-nAChR function are principally allosteric at both isoforms (rather than being dominated by direct interactions with ACh at the α4(+)/(−)α4-binding site, in the context of the LS isoform). This is not to imply that other parts of the α4(+)/(−)α4-binding site do not directly interact with ACh or other agonists; in fact our work and the work of others indicates that they do (35,–37).
The concatenated nAChR subunit approach offers substantial advantages in terms of expressing completely defined nAChR subtypes, including difficult-to-combine subunits (39, 40, 44). It also allows site-directed mutagenesis to be targeted to particular subunits when multiple copies are present in a pentameric complex (35,–37). As shown in Tables 11–3, functional parameters are very similar across all comparable nAChR compositions, whether expressed from linked or unlinked subunits. Critically, this validates the utility of concatemeric nAChR as a model in our studies of position-by-position subunit manipulation and our central discoveries revealed by systematic nAChR subunit interface alterations.
This study also illustrates the importance of measuring the effects of subunit mutation on cell surface expression of pure nAChR populations. Without the ability to normalize for cell surface nAChR expression, interpretation of differences between constructs in ion channel function per se would not be possible.
Although it is certainly true that precise interpretation of structure-activity relationships using only macroscopic CRCs can be problematic (48), previously published results allow us to make predictions. Changes in function per receptor must arise from altered single-channel conductance and/or open probability. A previous study of HS and LS isoform single-channel amplitudes indicates that the LS isoform may be associated with higher calculated chord conductance than the HS isoform (29 versus 21 picosiemens, respectively (22)). However, this difference is far less than required to account for the difference in function per receptor for these two isoforms as measured in this study (~10-fold greater for LS versus HS isoform α4β2-nAChR). Accordingly, we predict that the predominant effect of engaging the α4(+)/(−)α4 allosteric-like site is to increase channel-open probability. This would certainly be compatible with other prior findings. For example, it has been demonstrated that binding to the α4(+)/(−)α4 site is required for full agonist efficacy at LS isoform α4β2-nAChR (37). Furthermore, different parts of the same interface are known to form an allosteric Zn2+ potentiation site (49), and it has been shown that the principal effect of Zn2+ potentiation on α4β4-nAChR is to increase burst duration and therefore open probability (50). Therefore, it seems reasonable to hypothesize that altered open probabilities will also be the predominant factor driving the often large changes in per receptor function that are induced by incorporation of E-loop mutant subunits. This is especially so because E-loop mutations are located far from the channel domain; direct effects on channel conductance would not be anticipated.
Some generalizable features are revealed by viewing our findings first in terms of alterations in agonist-recognizing subunit interfaces. Nearly every time that an α4(+)/(−)β2 interface is converted to a mock α4(+)/(−)α4 interface, the result is an nAChR with LS-like features (EC50 value ≥60 μm). Conversely, there is never an LS profile for sensitivity to ACh when a natural α4(+)/(−)α4 interface is eliminated in the LSP backbone. These observations confirm and extend previous findings indicating that introduction of β2HQT subunits at α4(+)/(−)β2 interfaces makes them more closely resemble α4(+)/(−)α4 sites with lower ACh sensitivity (34, 38) and that α4(+)/(−)β2 interfaces contribute to higher agonist sensitivity. However, this approach is not sufficient to explain all of the findings of this study. As already discussed, introduction of an α4VFL mutant subunit anywhere in the LS isoform background (not just in the α4(+)/(−)α4 interface) produces a reduction in ACh EC50. In addition, as will be discussed next, this agonist-binding-site-only model is not adequate to explain several more observations.
Defining the precisely detailed mechanisms underlying differential α4(+)/(−)β2 agonist site contributions to HS and LS isoform α4β2-nAChR activation (such as changes in binding site affinity, interactions between the multiple binding sites present, and the extent of site-to-site cooperativity) is likely to require a sophisticated and extensive single-channel kinetics analysis, far beyond the scope of this study (48). However, the hypothesis that introducing mutations into the E-loops of α4β2-nAChR isoforms can significantly alter the allosteric activation mechanism of the host nAChR could also apply to β2HQT mutant subunits. In fact, this new hypothesis can explain our further otherwise-surprising observations (in addition to the position independence of the α4VFL subunit's effects).
In the first example, only the position 1 β2HQT mutant on an HSP backbone exhibits biphasic ACh activation (Figs. 2B and and3).3). This concatemer and the LSP construct are the only ones featuring an α4(+)/(−)α4-like agonist binding pocket flanked by unmutated β2 subunits (see inset to Fig. 3 for a circularized representation of the assembled position 1 β2HQT HSP construct). In the second example, a single β2HQT mutant-subunit substitution in either position 3 (where it does participate in an α4(+)/(−)β2 agonist binding pocket) or position 5 (where it does not participate in an agonist-binding site) has no effect on the resulting agonist CRCs (Fig. 2B). However, combining these two mutations, which are individually without effect, results in a substantial loss of agonist potency. This constitutes direct evidence that the E-loop status of a neighboring subunit can affect the functional contribution of a canonical α4(+)/(−)β2 agonist-binding site. It therefore appears that the functional effects of the β2HQT mutation are mediated allosterically in addition to its recently demonstrated effect on agonist binding potency (38). In the third example, and related to the preceding point, function per nAChR in the HSP β2HQT mutant series (Fig. 5) is significantly reduced only if both subunits at the β2(+)/(−)β2 interface are mutated (HSP β2HQTp1p5, and β2HQTp1p3p5). In addition, on the HSP backbone, the presence of just one α4(+)/(−)β2 interface is adequate to yield HS function except in the presence of β2HQTp5 altering the β2(+)/(−)β2 interface. These observations suggest that a wild-type β2(+)/(−)β2 interface is an important factor in HSP function. As a corollary to this observation, the LSP isoform, which lacks a β2(+)/(−)β2 interface, loses all HS component function with just one β2HQT substitution. Our results imply that E-loop allosteric effects constitute an important part of the context that results in otherwise identical α4(+)/(−)β2 agonist-binding sites contributing differentially to nAChR agonist activation both within HS isoform α4β2-nAChR and between the two α4β2-nAChR isoforms.
Interestingly, the current findings dovetail with and also help to explain observations in other studies (of α6β2*-nAChR) that pointed to the importance of α6 (−)-face E-loop effects on functional levels and agonist sensitivities (51,–53). In those publications, mouse (Asp-143 and Val-145) or human (Asn-143 and Met-145) α6 subunit residues determined in a species-specific manner functional effects of β3 subunit co-assembly into α6*-nAChR containing β2 or β4 subunits. These residues are nested between the α4 and β2 subunit E-loop residues that were targeted in this study. A very recent publication (54) also demonstrated interactions of an α4 (−)-face E-loop engineered into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein with a neighboring subunit. These interactions differed according to which particular ligand was bound (at least for compounds that are exceptionally selective between HS and LS phase activation), and appeared likely to impact receptor activation (54). If somewhat analogous interactions occur at interfaces not bound by agonist, this may provide at least a partial mechanistic basis for E-loop involvement in the neighbor effects noted in this study.
Our findings illustrate previously unappreciated complexity in the agonist activation mechanism of α4β2-nAChR, which are a predominant and physiologically important non-muscle nAChR subtype. It seems likely that similar phenomena may be seen in other Cys-loop receptors, given the high degree of structural similarity across members of the superfamily.
L. M. L. assisted with experimental design and interpretation, engineered the new concatenated nAChR constructs used in this study, performed electrophysiology experiments, and assisted with manuscript preparation (including figures and tables). M. M. W. and J. B. E. performed electrophysiology experiments and assisted with manuscript preparation (including figures and tables). J. F. C. produced 125I-mAb 295 and provided essential guidance on experimental design. J. M. L. assisted with manuscript drafting and provided critical revision of its content. R. J. L. assisted with experimental design and interpretation and with manuscript writing and revisions. P. W. conceived and coordinated the study, performed the 125I-mAb 295 binding experiments, and (with advice and counsel from the other authors) was principally responsible for data interpretation and manuscript writing. All authors reviewed the results and approved the final version of the manuscript.
We thank Dr. Isabel Bermudez (Oxford Brookes University, Oxford, UK) for providing the original HSP and LSP α4β2 constructs that were modified for use in this study and for extensive advice on their use. We also thank Drs. F. Ivy Carroll (Research Triangle Institute, Research Triangle Park, NC) and Alan P. Kozikowski (University of Illinois, Chicago) for gifts of compounds.
*This work was supported by National Institutes of Health Grants DA026627 and DA012242 (to P. W.) and DA030929 (to J. M. L.) and by endowment and capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation (to P. W. and R. J. L.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
2The abbreviations used are: