Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

Background: Naturally expressed mouse (m) α6*-nAChRs have negligible functional expression in vitro. Results: Functional expression of mouse α6β2β3- or α6β4β3-nAChRs is enhanced upon manipulation of β3 subunit N-terminal extracellular domain residues. Conclusion: N-terminal extracellular domains in nAChR β3 subunits play heretofore underappreciated roles in controlling functional expression of α6*-nAChR. Significance: nAChR “accessory” subunits are critical elements in nAChR assembly and function. Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs.

Here we report that nAChR h␤3 or h␤3 V9ЈS (i.e. h␤3(V273S)) subunits coexpressed in oocytes also expressing m␣6 subunits in the presence of m␤4 or m␤2 subunits yielded nAChRs with higher levels of function than those of m␣6m␤4m␤3-or m␣6m␤2m␤3 V9ЈS -nAChRs. Further studies using chimeric or gain-of-function chimeric mouse/human nAChR ␤3 subunits and site-directed mutagenesis identified AA residues in the extracellular N-terminal domain (NTD; in so-called loops "␤2-␤3," C, and E) of m␤3 subunits that when substituted with corresponding residues from h␤3 subunits alone or in some spe-cific combinations increased the function of m␣6m␤2*-and m␣6m␤4*-nAChRs. These studies elucidate some of the structural bases dictating roles for nAChR ␤3 subunits in functional expression of m␣6m␤2*-and m␣6m␤4*-nAChRs.

EXPERIMENTAL PROCEDURES
Bioinformatics and Homology Modeling-Using several Web-available threading methods, the ␤1 subunit of the muscle nicotinic acetylcholine receptor of the marbled electric ray (Torpedo marmorata) (2BG9.B; Protein Data Bank code 2BG9 Chain B) (35) was identified as a suitable template for threedimensional modeling of m␤3 subunits (SWISS-MODEL Protein Modeling Server) (36). The overall stereochemical quality of the final model was assessed by the program PROCHECK (37). The homology model for the nAChR m␤3 subunit was rendered using UCSF Chimera, a program for interactive visualization and analysis of molecular structures. Protein sequences for nAChR ␤3 subunits of several species or mouse nAChR subunits retrieved from the National Center for Biotechnology Information (NCBI) Entrez Web service were aligned with each other using the Web program ClustalW.
Chemicals-All chemicals for electrophysiology were obtained from Sigma. Fresh agonist (acetylcholine (ACh) or nicotine) or antagonist (atropine) stock solutions were made daily or diluted from frozen stock in Ringer's solution (OR2), which consisted of 92.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES, pH 7.5.
nAChR ␤3 Subunit Chimeras-Guided by an alignment of nAChR h␤3 and m␤3 subunit protein sequences (see Fig. 1A), chimeric mouse/human ␤3 subunits were designed and created (see Fig. 1B) as described below. We cared to construct chimeras in a manner that isolated domains and/or structural features in ␤3 subunits.
Construction of the m␤3(1-187)/h␤3(182-458) Chimeric Subunit (SalI Site-based Construct)-Mouse nAChR ␤3 subunits possess an innate SalI restriction site (see Fig. 1) in the NTD around AA residue Val 187 . A SalI restriction site (Table 1)   TABLE 1 Primers and restriction sites used to create mutant/chimeric constructs For mutants, the first amino acid (single letter code; numbering begins at the translation start methionine) designates the wild-type nAChR (human or mouse) subunit residue that is replaced with the indicated second amino acid. In the forward primer nucleic acid sequence, capitalization indicates the nucleotide(s) changed from the wild-type subunit to create the corresponding mutant or restriction site.
Preparation of cRNA Mixtures for Injection-We planned to introduce identical amounts of cRNA, presumably producing equal amounts of each subunit protein, into oocytes largely due to lack of information about the levels of mRNA for each subunit that composes ␣6*-nAChRs in neurons or cells. We provisionally assumed that ␣6 subunits or their mutants in association with ␤2 or ␤4 subunits would form complexes having 2:3 and/or 3:2 ratios of the indicated subunits and that oocytes also injected with WT, chimeric, or other forms of ␤3 subunits would express nAChR with 2:2:1 ratios of ␣:␤:␤3 subunits. For expression of binary nAChRs (i.e. nAChRs containing two subunits; ␣ ϩ ␤ but not ␤3), cRNA mixtures were prepared by mixing 1 l of cRNA for each subunit and an additional microliter of RNase-free water (i.e. total volume, 3 l). Similarly, for expression of trinary nAChRs (i.e. nAChRs containing three subunits; (␣ ϩ ␤) ϩ ␤3) cRNA mixtures were prepared by mixing 1 l of cRNA for each subunit. Several preparations of each cRNA mixture were prepared and stored at Ϫ80°C until further use.
cRNA concentrations for each nAChR ␣ and ␤ subunit were adjusted to 150 ng/l for the first set of experiments (for data presented in Table 2 and Fig. 2). As noted above, introduction of 69 nl of cRNAs (from a 3-l cRNA mixture) into each oocyte would deliver ϳ3.5 ng of cRNA for each ␣ and ␤ subunit whether binary or trinary nAChRs are expressed. For all other experiments, concentrations of cRNAs prepared for each nAChR ␣ and ␤ subunit were adjusted to 500 ng l Ϫ1 . Injection of 138 nl of cRNA of a 3-l cRNA mixture into each oocyte would deliver ϳ23 ng of cRNAs for each ␣ and ␤ subunit whether binary or trinary nAChRs are expressed.
Oocyte Preparation and cRNA Injection-Female Xenopus laevis (Xenopus I, Ann Arbor, MI or Nasco, Fort Atkinson, WI) were anesthetized using 0.2% tricaine methanesulfonate (MS-222) (Sigma or Nasco). Ovarian lobes were surgically removed from the frogs and placed in an incubation solution that consisted of 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM Na 2 HPO 4 , 0.6 mM theophylline, 2.5 mM sodium pyruvate, 5 mM HEPES, 50 mg/ml gentamycin, 50 units/ml penicillin, and 50 g/ml streptomycin, pH 7.5. The lobes were cut into small pieces and digested with 0.08 Wünsch units/ml Liberase Blendzyme 3 (Roche Applied Science) with constant stirring at room FIGURE 1. Construction of nAChR ␤3 subunit chimeras. A, alignment of nAChR h␤3 and m␤3 subunit protein sequences. Amino acid sequences of nAChR h␤3 (accession number NP_000740.1) and m␤3 subunits (accession number AAL75573.1) retrieved from GenBank TM were aligned using protein BLAST. AA numbering begins at the translation start methionine. Identical residues between human and mouse nAChR ␤3 subunits are indicated by a dash (-). Putative loop regions (A, B, and C in the primary face and D, E, and F in the complementary face), TM domains (I, II, III, and IV), and TM II 9Ј and 13Ј amino acid residues in human and mouse nAChR ␤3 subunits are identified. nAChR m␤3 subunit residues (Gln 94 , Glu 101 , Asn 107 , Ser 144 , Ser 148 , Asp 221 , and Phe 223 ) given prime attention in mutagenesis studies are underlined and in boldface. An upward arrow (1) indicates junctions for chimeric subunits and the restriction sites used for construction of chimeric mouse/human ␤3 subunits. Colons (:) below sequences indicate conserved residues. B, schematic diagrams of chimeric nAChR ␤3 subunits. Chimeric mouse/human nAChR ␤3 subunits with or without V9ЈS mutations in their respective TM II were constructed. The 273rd AA in nAChR h␤3 subunit or the 279th AA in nAChR m␤3 subunit is a valine and is noted as the 9Ј AA in TM II. N, N-terminal domain; I, II, III, or IV, respective TM domains; cyto-loop, cytoplasmic loop; C, C terminus. Numbers in brackets are the regions of the indicated subunits that are used for construction of mouse/human ␤3 chimeric subunits. temperature for 1.5-2 h. The dispersed oocytes were thoroughly rinsed with incubation solution. Stage VI oocytes were selected and incubated at 16°C before injection. Micropipettes used for injection were pulled from borosilicate glass (Drummond Scientific, Broomall, PA) using a Sutter P87 or P1000 horizontal puller (Sutter Instrument Co., Novato, CA), and the tips were broken with forceps to ϳ40 m in diameter. cRNAs were drawn up into the micropipette and injected into oocytes using a Nanoject or Nanoject II microinjection system (Drummond Scientific) at a total volume of 69 or 138 nl.
Oocyte Electrophysiology-Two to 5 days after injection, oocytes were placed in a small volume chamber and continuously perfused with OR2. The chamber was grounded through an agarose bridge. The oocytes were voltage-clamped at Ϫ70 mV (unless otherwise noted) to measure agonist-or antagonist-induced currents using Axoclamp 900A and pClamp 10.2 software (Axon Instruments/Molecular Devices, Sunnyvale, CA). The current signal was low pass-filtered at 10 Hz with the built-in low pass Bessel filter in the Axoclamp 900A and digitized at 20 Hz with Axon Digidata1440A and pClamp10. Electrodes contained 3 M KCl and had a resistance of 1-2 megaohms. Drugs (agonists and antagonists) were prepared daily in bath solution. Drug was applied using a Valvelink 8.2 perfusion system (AutoMate Scientific, Berkeley, CA). Atropine (1 M) was always co-applied for ACh-based recordings to eliminate muscarinic acetylcholine receptor responses. nAChR ␤3 constructs were tested individually or in batches as they became available to get an estimate of their effect on the function of ␣6*-nAChRs. Then, for the purpose of comparison, electrophysiological recordings were performed in a given day in a given batch of oocytes following the same order of injections. Hence data points in a figure panel were obtained under similar experimental conditions.
Experimental Controls-Injection of water or empty vector (used as two forms of negative controls) or of cRNA corresponding to one subunit alone or pairwise combinations of nAChR ␤3, ␤3 V9ЈS , ␤3 V13ЈS , or other forms of ␤3 subunits with either an ␣6 or other forms of ␣6 or ␤4 subunit (ϳ7-46 ng of total cRNA) did not result in the expression of functional nAChRs. Current responses to 100 M nicotine or ACh were less than 5-10 nA (data not shown).
Data Analyses-Raw data were collected and processed in part using pClamp 10.2 (Molecular Devices) and a spreadsheet (Excel, Microsoft, Bellevue, WA) using peak current amplitudes as measures of functional nAChR expression, and results were pooled across experiments (mean Ϯ S.E. for data from at least three oocytes). In some cases, mean peak current amplitudes in response to a single concentration of an agonist were compared across different subunit combinations. However, assessment of true I max values for different nAChR subunit combinations required assessment based on more complete concentration-response relationships in which mean peak current amplitudes at specified ligand concentrations were fit to the Hill equation or its variants using Prism 4 (GraphPad Software, San Diego, CA). F-tests (p Ͻ 0.05 to define statistical significance) were carried out to compare the best fit values of log molar EC 50 values across specific nAChR subunit combinations.
There are limitations in the ability to compare levels of functional nAChR expression even though we injected similar amounts of RNA for all constructs. This is because expression levels assessed as peak current amplitudes are affected by batch-to-batch variation in oocytes, time between cRNA injection and recording, and subunit combination-specific parameters, such as open probability (influenced by gating rate constants and rates and extents of desensitization), single channel conductance, assembly efficiency, and efficiency of receptor trafficking to the cell surface (39). We made no attempt to measure or control for subunit combination-specific effects, but whenever preliminary studies revealed possible differences in peak current amplitudes, findings were further confirmed across different subunit combinations using the same batch of oocytes and the same time between cRNA injection and recording (15,19,30,38). Therefore peak current amplitudes shown for representative traces in some figures, pooled data from limited sets of studies, and mean peak current amplitudes across all studies for a given combination of subunits given in tables or figures sometimes differ. However, when we make statements about results comparing ligand potencies and efficacies across subunit combinations, the observations are clear, significant, and in agreement whether for pooled data or for results from smaller sets of studies (one-way analyses of variance followed by Tukey's multiple comparison tests).

RESULTS
Previously (15) we have shown that coexpression of WT nAChR ␣6 and ␤2 subunits alone or in combination with ␤3 or ␤3 V9ЈS subunits in oocytes, all from a single species (human or mouse), did not yield consistent and reproducible current responses to nicotinic agonists. However, under similar experimental conditions, we were able to show that coexpression of m␣6 subunits, instead of h␣6 subunits, with h␤2 and h␤3 V9ЈS subunits led to expression of functional hybrid m␣6h␤2h␤3 V9ЈS -nAChRs (15). Also, hybrid m␣6h␤4h␤3-nAChRs were fully functional, although there was no function for h␣6h␤4h␤3or m␣6m␤4m␤3-nAChRs (15). These studies were carried out by injecting ϳ1-6 ng of cRNAs for each subunit into oocytes. In continuation of our earlier efforts, in this study, we substituted human ␤3 subunits for mouse ␤3 subunits. Initially we injected about ϳ3.5 ng of cRNAs for each nAChR subunit to express hybrid nAChRs, but later we increased amounts injected to ϳ23 ng for each subunit to emulate the approach taken by Kuryatov et al. (14) to express functional human ␣6␤4*-nAChRs.
Incorporation of nAChR h␤3, h␤3 V9ЈS , or h␤3 V13ЈS Subunits Potentiates m␣6m␤4*-nAChR Function-Coexpression with WT h␤3 subunits significantly (p Ͻ 0.05) potentiated ACh-or nicotine-induced current responses of m␣6m␤4-nAChRs ( Fig.  2 and Table 2). Also, coexpression with nAChR h␤3 V9ЈS or h␤3 V13ЈS subunits increased (p Ͻ 0.05) the current responses further ( Fig. 2 and Table 2). The increase in agonist sensitivities and in peak current amplitudes indicate that WT h␤3 subunits incorporate into at least some complexes containing m␣6 and m␤4 subunits and these effects are most likely due to higher levels of functional receptor expression. Moreover, whereas not all oocytes expressing m␣6 and m␤4 subunits yield functional responses to nicotinic agonists, almost all oocytes expressing nAChR m␣6, m␤4, and h␤3 subunits produced functional responses, suggesting that nAChR h␤3 subunits facilitate formation of functional, trinary (containing three kinds of subunits) nAChRs.
Spontaneously Opening m␣6m␤4h␤3 V9ЈS -or m␣6m␤4h␤3 V13ЈS -nAChRs Are Sensitive to Blockade by Atropine-Atropine (1 M) was always co-applied for ACh-based recordings to eliminate muscarinic acetylcholine receptor responses. Because atropine at higher concentrations also can interact with different nAChR subtypes (15,40,41), initially as a simple control, we assessed the effects of atropine at different concentrations alone on all receptor combinations studied. Atropine alone did not produce any effect when assessed using oocytes expressing any combination of WT nAChR subunits (data not shown), but it reversibly produced outward currents when applied to oocytes expressing receptors containing ␤3 V9ЈS or ␤3 V13ЈS subunits. The concentration-dependent effects of atropine were Leftward shifts in agonist concentration-response curves are evident for functional nAChR containing h␤3, h␤3 V9ЈS , or h␤3 V13ЈS subunits (p Ͻ 0.0001; ϳ2-, ϳ717-, and ϳ418-fold, respectively, for ACh EC 50 values and ϳ6-, ϳ605-, and 217-fold, respectively, for nicotine EC 50 values relative to the respective agonist EC 50 values for activation of m␣6m␤4-nAChR function). See Table 2 for parameters.

TABLE 2
Parameters for drug action at hybrid h␣6h␤4*-or m␣6m␤4*-nAChRs Potencies (micromolar EC 50 or IC 50 values with 95% confidence intervals (CI)), Hill coefficients (n H Ϯ S.E.), mean Ϯ S.E. efficacies (I max in nA), and concentrations (conc.) where maximal peak current amplitudes (I max ) are achieved (M) are provided for the indicated agonist (ACh or nicotine) or antagonist (atropine) acting at nAChR composed of the indicated subunits derived from the specified species and from the indicated number of independent experiments (n) based on studies as shown in Fig. 2. 1 indicates a significant (p Ͻ 0.05) increase in potency or efficacy of the specified agonist at the indicated nAChR subtype relative to nAChR upon expression in the presence of the indicated wild-type or mutant ␤3 subunit instead of in the absence of a ␤3 subunit, OE indicates a significant increase in specified agonist potency or efficacy at the indicated nAChR subtype upon substitution of a mutant for a wild-type ␤3 subunit relative to nAChR containing the same subunits in the presence of wild-type ␤3 subunits, and ‚ or ƒ indicates a significant increase or decrease, respectively, in potency or efficacy of the specified agonist or antagonist at the indicated nAChR subtype containing ␤3 V13ЈS instead of ␤3 V9ЈS subunits.  2 and Table 2). It is estimated, based on comparisons of atropine-induced outward current peak amplitudes with the sum of those currents plus inward currents induced by fully efficacious concentrations of nicotine or ACh, that more than 4 -16% of these receptors are spontaneously open at any one time.
N-terminal AA Residues in the nAChR m␤3 Subunit That Influence the Function of Mouse ␣6␤2*and ␣6␤4*-nAChRs-Because effects on m␣6m␤2*and m␣6m␤4*-nAChR function were most extreme in chimeras containing extracellular N-terminal domains from the m␤3 subunit, we focused on this region and on residues that differ between h␤3 and m␤3 subunits. For all nAChR subunits, there is a "primary" or (ϩ) face and a "complementary" or (Ϫ) face where subunit extracellular N-terminal domains interact, forming a subunit interface. Interface interactions are critical for subunits to form dimers and for dimers to join with a single subunit to close the penta-meric assembly. Interfaces involving the primary face of specific ␣ subunits and the complementary face of specific ␤ subunits also are known to contain agonist binding pockets, occupancy of which leads to channel opening and where competitive antagonists also bind to affect function (the ␣ subunit was designated as that providing the primary face because it was initially thought that agonist binding sites resided solely within ␣ subunits). nAChR biologists have identified several loops at turns in ␤-strands that criss-cross subunit extracellular domains as in a woven basket. So-called loop ␤2-␤3 (named so because the loop is formed at the tip of a turn between ␤ strands ␤2 and ␤3) and loops D, E, and F are evident from modeling and structural studies to be on the complementary face of a given subunit, whereas loops A, B, and C are on the primary face. Loops A-F appear to be engaged in ligand recognition. For site-directed mutagenesis studies, we focused on some of the very few residues that differ between h␤3 and m␤3 subunits, AAs Gln 94 , Glu 101 , and Asn 107 in the ␤2-␤3 loop; AAs Ser 144 and Ser 148 in putative loop E, and AAs Glu 221 and Phe 223 in putative loop C, to determine roles in functional expression of m␣6m␤2*and m␣6m␤4*-nAChRs (Fig. 1A). Residues in the nAChR m␤3 subunit were mutated to their counterparts in the nAChR h␤3 subunit alone or in specific combinations (i.e. m␤3(Q94H), m␤3(E101D), m␤3(N107H), m␤3(Q94H/ E101D), m␤3(Q94H/N107H), m␤3(S144N/S148V), m␤3(E221D), and m␤3(E221D/F223V)) (Fig. 5).

TABLE 3 Parameters for nicotine action at nAChRs containing m␣6 and m␤3 mutant subunits
Potencies (micromolar EC 50 values with 95% confidence intervals (CI)), Hill coefficients (n H Ϯ S.E.), mean Ϯ S.E. efficacies (I max in nA), and concentrations (conc.) where maximal peak current amplitudes (I max ) are achieved (M) are provided for nicotine acting at mouse nAChR composed of the indicated subunits and from the indicated number of independent experiments (n) based on studies as shown in Fig. 6. 1 indicates a significant (p Ͻ 0.05) increase in the indicated parameter at the indicated nAChR subtype relative to wild-type m␣6m␤4-nAChR, and OE indicates a significant (p Ͻ 0.05) increase in the indicated parameter at the indicated nAChR subtype relative to wild-type m␣6m␤4m␤3-nAChR.

Potency
Peak than those expressing m␣6 V13ЈS m␤2m␤3-nAChRs. These results confirm the previous findings that TM II 13Ј valine-toserine mutations in the m␣6 subunit are more capable of attributing gain of function to m␣6*-nAChRs than the TM II 9Ј leucine-to-serine mutation (38).
We noticed that upon substitution of nAChR h␤3 subunits for nAChR m␤3 subunits highly functional hybrid m␣6m␤4h␤3-nAChRs were produced in oocytes. We also noticed that functional m␣6m␤4m␤3-nAChRs were formed in oocytes when they were expressed using an injection of relatively larger amounts of cRNAs (ϳ23 ng) for each subunit. The peak current responses of these m␣6m␤4m␤3-nAChRs were nonetheless severalfold lower than those of hybrid m␣6m␤4h␤3-nAChRs. Similar to previous observations (15), whether using relatively lower or higher amounts of injected cRNA for each subunit, functional m␣6m␤2-, m␣6m␤2m␤3-, or m␣6m␤2h␤3-nAChRs were not detected in oocytes. However, upon increasing the amount of cRNA injected for each subunit, minimally functional m␣6m␤2m␤3 V9ЈS -or robustly functional m␣6m␤2h␤3 V9ЈS -nAChRs emerged on cell surfaces. Additionally, m␣6m␤4*-nAChRs harboring h␤3 V9ЈS or h␤3 V13ЈS subunits showed gain of function similar to those of m␣6m␤4m␤3 V9ЈS -or m␣6m␤4m␤3 V13ЈS -nAChRs (15). Therefore, incorporation of h␤3 or m␤3 subunits into m␣6m␤4*-nAChRs is evident because it had a potentiation effect. These results also suggest that these WT ␤3 subunits must be facilitating assembly of functional receptors. Potentiation of agonist sensitivity and levels of functional responses also indicate that there was incorporation of mutant h␤3 V9ЈS or m␤3 V9ЈS subunits into m␣6m␤4*or m␣6m␤2*-nAChRs with further facilitation of functional receptor expression, increased frequency of agonist-gated channel opening, or both. These results also are indicative of efficient incorporation of h␤3 subunits, but not that of m␤3 subunits, into assemblies of m␣6 and m␤4 subunits or of m␣6 and m␤2 subunits.
Differences in amino acid composition between h␤3 and m␤3 subunit extracellular N-terminal and second cytoplasmic loop regions (e.g. as opposed to their nearly identical transmembrane domains) that influence effects on ␣6*-nAChR function were revealed based on studies of chimeric nAChR mouse/human ␤3 subunits or their gain-of-function variants. The involvement of the NTD of ␤3 subunits in these effects echoes previous findings that the NTD of h␣6 subunits influences assembly and function of human ␣6␤3*-nAChRs (15,19,38).
The current site-directed mutagenesis studies indicate that substitution of m␤3 subunit AA residues in primary face loop C with h␤3 subunit residues enhanced functional expression of m␣6m␤2m␤3 V9ЈS -nAChRs. In addition, h␤3 subunit AA substitutions in complementary face ␤2-␤3 and E loops for residues in m␤3 subunits increased functional expression of m␣6m␤4m␤3-nAChRs. These results are in agreement with the previous observations that substitutions at extracellular N-terminal loops influence functional expression of h␣6*-nAChRs and other subtypes of nAChRs (15,19,38,47).
The increased functional expression of m␣6*-nAChRs seen upon AA substitution in m␤3 subunits must be due to some combination of increases in efficiency of incorporation of subunits into receptor complexes, trafficking to the cell surface, and/or preservation of cell surface receptors. nAChR m␤3 subunit loop E residues Ser 144 and Ser 148 differ from Asn or Val residues, respectively, in h␤3 subunits in side chain length and possibility of engaging in glycosylation (Ser versus Asn) and hydrophobicity (Ser versus Val) (48). nAChR m␤3 subunit loop C residues Glu 221 and Phe 223 differ from Asp or Val AAs in h␤3 subunits in side chain length (Glu versus Asp and Phe versus Val) and to some degree in hydrophobicity (Phe versus Val). These differences in AAs could influence interactions with adjacent (or distant?) ␤2, ␤4, or ␣6 subunits that are important for m␣6m␤2*and m␣6m␤4*-nAChR assembly (Fig. 8D).
␤2-␤3 or E loop residues in the negative (Ϫ) or complementary face of the m␤3 subunit would be involved in presumed interactions with residues on the positive (ϩ) or primary faces of the neighboring m␤4 or m␤2 subunit, and loop C residues in the positive (ϩ) or primary face of the ␤3 subunit would be involved in presumed interactions with residues on the negative (Ϫ) or complementary faces of the neighboring m␣6 subunit in a complex that has the presumed arrangement of Ϫ (␤3 or ␤3 V9ЈS ) ϩ : Ϫ ␣6 ϩ : Ϫ (␤2 or ␤4) ϩ : Ϫ ␣6 ϩ : Ϫ (␤2 or ␤4) ϩ where ligand binding pockets are thought to be located between the primary (ϩ) face of m␣6 and complementary (Ϫ) face of the m␤2 or m␤4 subunits (i.e. ␣6 ϩ : Ϫ ␤4 or ␣6 ϩ : Ϫ ␤2) (Fig. 8). Agonist binding is not expected occur at ␤3 ϩ : Ϫ (␤2 or ␤4) or ␤3 ϩ : Ϫ ␣6 subunit interfaces. However, recent evidence suggests that interfaces involving subunits in the accessory subunit position where the ␤3 subunit would be situated can engage in allosteric or co-agonist effects (49 -51). Residues in or equivalent to those at m␤3 subunit positions 94, 101, 107, 144, 148, 221, and 223 are FIGURE 8. Illustration of nAChR ␤3 subunit residues and its interfaces that are important in the function of m␣6*-nAChRs. A, sequence alignment of nAChR ␤3 subunit proteins from several species. nAChR ␤3 protein sequences extracted from GenBank accession numbers NP_ 775304.1 (Mouse; Mus musculus), NP_000740.1 (Human; Homo sapiens), NP_990143.1 (Chicken; Gallus gallus), NP_001080652.1 (Frog; X. laevis), NP_598281.1 (Rat; Rattus norvegicus), NP_001029105.1 (Chimpanzee; Pan troglodytes), XP_599970.2 (Cow; Bos taurus), and NP_775394.1 (Zebrafish; Danio rerio) were aligned using ClustalW. B, sequence alignment of mouse nAChR ␤3 subunit proteins. Mouse nAChR subunits were aligned using ClustalW. For both A and B, numbering begins at the translation start methionine of the mouse nAChR ␤3 subunit protein and is shown in the N-terminal domain region of interest. Symbols below sequences indicate fully (*), strongly (:), or weakly (.) conserved residues, and underlining in shaded face indicates numbered residues in nAChR m␤3 subunit targeted for mutagenesis studies. C, a three-dimensional model of the N-terminal domain of mouse nAChR ␤3 subunit. A three-dimensional model of the mouse nAChR ␤3 subunit was generated based on the crystal structure of Torpedo muscle nAChR ␤ subunit (Protein Data Bank code 2BG9:B). The N-terminal domain of the nAChR m␤3 subunit possesses ␤ strands that form a ␤ sandwich and conforms to an immunoglobulin fold. AA residues in the ␤2-␤3 loop (Gln 94 and Glu 101 ), loop E (Ser 144 and Ser 148 ), or loop C (Glu 221 and Phe 223 ) that positively influence the current responses of m␣6*-nAChRs are identified. The figure displayed was drawn using the program Chimera. D, schematic illustration of the composition of m␣m␤4*-nAChRs. Adhering to the canonical rule of pentamer formation, m␣6m␤4-nAChRs would be formed of three ␣6 and two ␤4 subunits (left) or two ␣6 and three ␤4 subunits (middle). In the event m␤3 subunits are integrated into m␣6m␤4*-nAChRs, they would substitute for the third m␣6 subunit in the first (left) configuration or the third m␤4 subunit in the second (middle) configuration, occupying what is labeled as the fifth position (yellow). Agonist (ACh or nicotine and others) binding sites at the interfaces between ␣6 and either ␤2 or ␤4 subunits are shown as ovals. Results from the current study (right) support the idea that the ␤2-␤3 loop and loop E residues in the (Ϫ) face and/or loop C residues in the (ϩ) face (arrows) of the m␤3 subunit are important in higher functional expression of m␣6m␤4*-nAChRs. Mouse ␣6␤2*-nAChRs would attain similar configurations, but the ␤2 subunit would substitute for the ␤4 subunit.
Effects of ␤3 Subunits on Mouse ␣6*-nAChR Function OCTOBER 10, 2014 • VOLUME 289 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28349 conserved with those in rats but differ from those that are conserved within primates (human and chimp; Fig. 8A). These m␤3 subunit AAs also are unique across mouse nAChR subunits (Fig. 8B). We have advanced the possibility that these residues could affect efficiency of ␣6*-nAChR assembly (not altering agonist potency but affecting peak current responses as for m␣6*-nAChRs harboring m␤3(S144N/S148V), m␤3(E221D), or m␤3(E221D/F223V) subunits). Another intriguing possibility is that these unique residues could allow formation of novel classes of ligand binding sites at ␤2/␤4:␤3 or ␤3:␣6 subunit interfaces that also could lead to changes in levels of receptor function as for ligand occupancy of the ␣4:␣4 subunit interface in low sensitivity ␣4␤2*-nAChR (51).
Plenty of information is available on the role of primary face loops (A, B, and C) from ␣ subunits and complementary face loops (D, E, and F) from ␤ subunits that participate in ligand binding largely from structural and/or mutagenesis studies of muscle-type, ␣7-, or other nAChRs and from lower eukaryotic and prokaryotic proteins structurally homologous to the extracellular domain of nAChRs (52)(53)(54). Our results presented here, for the first time, show that extracellular N-terminal domain loops of the accessory subunit, ␤3, regulate the functional expression of m␣6*-nAChRs. These results also provide further evidence that nAChR ␤3 subunits not only form functional receptors in combination with nAChR ␣6 subunits but also can enhance their function by interacting with adjacent subunits mediated by N-terminal loop residues. Current findings lay a foundation for enhanced functional expression of m␣6*-nAChRs that could facilitate the discovery and development of nicotinic ligands that selectively interact with ␣6*-nAChRs. These results could be useful to fuel and inform emerging interest in ␣6 and ␤3 subunits and the receptors they compose with specific reference to possible roles in locomotion, reward and reinforcement behavior, schizophrenia, and Parkinson disease (5,6,55).