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Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant*

Open AccessPublished:June 04, 2012DOI:https://doi.org/10.1074/jbc.M112.379339
      Background: The naturally occurring α5(D398N) variant alters smoking behavior, but functional differences have not been detected between α3β4α5 nAChR harboring these variants.
      Results: ACh-induced α3β4α5 nAChR function is lower when α5(Asn-398) substitutes for α5(Asp-398).
      Conclusion: The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.
      Significance: α3β4α5 nAChR function may be a useful target for smoking cessation pharmacotherapies.

      Introduction

      Nicotinic acetylcholine receptors (nAChR)
      The abbreviations used are: nAChR
      nicotinic acetylcholine receptor(s)
      ACh
      acetylcholine
      CI
      confidence interval
      ANOVA
      analysis of variance.
      are prototypical members of the ligand-gated ion channel superfamily of neurotransmitter receptors. nAChR exist as a diverse family of molecules composed of different pentameric combinations of homologous subunits derived from at least 17 genes (α1-α10, β1-β4, γ, δ, ϵ). The properties of nAChR are determined by their subunit composition, giving rise to multiple subtypes with a range of overlapping pharmacological and biophysical properties (
      • Gotti C.
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      • Gaimarri A.
      • Guiducci S.
      • Manfredi I.
      • Moretti M.
      • Pedrazzi P.
      • Pucci L.
      • Zoli M.
      Structural and functional diversity of native brain neuronal nicotinic receptors.
      ). It also has become apparent that different stoichiometries of the same subunits can produce subtypes with distinctly different characteristics, a phenomenon observed in both heterologous and natural expression systems (
      • Gotti C.
      • Clementi F.
      • Fornari A.
      • Gaimarri A.
      • Guiducci S.
      • Manfredi I.
      • Moretti M.
      • Pedrazzi P.
      • Pucci L.
      • Zoli M.
      Structural and functional diversity of native brain neuronal nicotinic receptors.
      ,
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      Four pharmacologically distinct subtypes of α4β2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.
      ,
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      ,
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      Human α4β2 acetylcholine receptors formed from linked subunits.
      ,
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      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ).
      Recently, genome-wide association studies have indicated that single-nucleotide polymorphisms (SNPs) within nAChR subunits can substantially affect nAChR-mediated smoking behavior in humans. Most prominent among these single-nucleotide polymorphisms have been those located in the CHRNA5/CHRNA3/CHRNB4 locus, located on chromosome 15q25, which encodes the α5, α3 and β4 subunits of nicotinic receptors. This locus was first associated with nicotine dependence (
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      A variant associated with nicotine dependence, lung cancer and peripheral arterial disease.
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      Variants in nicotinic receptors and risk for nicotine dependence.
      ). One non-synonymous polymorphism (rs16969968), which changes the 398th amino acid from aspartic acid to asparagine (D398N) in the α5 subunit, is particularly strongly associated with greater risk for increased nicotine consumption. Interestingly, variants at this locus also are associated with increased liability for lung cancer (
      • Thorgeirsson T.E.
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      • Gudbjartsson T.
      • Jones G.T.
      • Mueller T.
      • Gottsäter A.
      • Flex A.
      • Aben K.K.
      • de Vegt F.
      • Mulders P.F.
      • Isla D.
      • Vidal M.J.
      • Asin L.
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      • Murillo L.
      • Blondal T.
      • Kolbeinsson H.
      • Stefansson J.G.
      • Hansdottir I.
      • Runarsdottir V.
      • Pola R.
      • Lindblad B.
      • van Rij A.M.
      • Dieplinger B.
      • Haltmayer M.
      • Mayordomo J.I.
      • Kiemeney L.A.
      • Matthiasson S.E.
      • Oskarsson H.
      • Tyrfingsson T.
      • Gudbjartsson D.F.
      • Gulcher J.R.
      • Jonsson S.
      • Thorsteinsdottir U.
      • Kong A.
      • Stefansson K.
      A variant associated with nicotine dependence, lung cancer and peripheral arterial disease.
      ,
      • Spitz M.R.
      • Amos C.I.
      • Dong Q.
      • Lin J.
      • Wu X.
      The CHRNA5-A3 region on chromosome 15q24–25.1 is a risk factor both for nicotine dependence and for lung cancer.
      ,
      • Hung R.J.
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      • Mukeria A.
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      • Lathrop M.
      • Brennan P.
      A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25.
      ), and possibly with decreased risk for alcoholism (
      • Schlaepfer I.R.
      • Hoft N.R.
      • Collins A.C.
      • Corley R.P.
      • Hewitt J.K.
      • Hopfer C.J.
      • Lessem J.M.
      • McQueen M.B.
      • Rhee S.H.
      • Ehringer M.A.
      The CHRNA5/A3/B4 gene cluster variability as an important determinant of early alcohol and tobacco initiation in young adults.
      ,
      • Wang J.C.
      • Grucza R.
      • Cruchaga C.
      • Hinrichs A.L.
      • Bertelsen S.
      • Budde J.P.
      • Fox L.
      • Goldstein E.
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      • Edenberg H.J.
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      • Goate A.M.
      Genetic variation in the CHRNA5 gene affects mRNA levels and is associated with risk for alcohol dependence.
      ) and cocaine dependence (
      • Grucza R.A.
      • Wang J.C.
      • Stitzel J.A.
      • Hinrichs A.L.
      • Saccone S.F.
      • Saccone N.L.
      • Bucholz K.K.
      • Cloninger C.R.
      • Neuman R.J.
      • Budde J.P.
      • Fox L.
      • Bertelsen S.
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      • Almasy L.
      • Porjesz B.
      • Kuperman S.
      • Schuckit M.A.
      • Edenberg H.J.
      • Rice J.P.
      • Goate A.M.
      • Bierut L.J.
      A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence.
      ).
      These observations raise the question of what the functional effects of the D398N mutation might be. The α5 subunit can only assemble into functional nAChR when expressed with at least two other subunits (
      • Gotti C.
      • Clementi F.
      • Fornari A.
      • Gaimarri A.
      • Guiducci S.
      • Manfredi I.
      • Moretti M.
      • Pedrazzi P.
      • Pucci L.
      • Zoli M.
      Structural and functional diversity of native brain neuronal nicotinic receptors.
      ). In the central nervous system, most α5 subunit expression occurs in combination with α4 and β2 subunits (
      • Brown R.W.
      • Collins A.C.
      • Lindstrom J.M.
      • Whiteaker P.
      Nicotinic α5 subunit deletion locally reduces high affinity agonist activation without altering nicotinic receptor numbers.
      ,
      • Mao D.
      • Perry D.C.
      • Yasuda R.P.
      • Wolfe B.B.
      • Kellar K.J.
      The α4β2α5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo.
      ). Experiments using heterologous expression systems have demonstrated that α4β2* nAChR containing α5 subunits harboring the risk (Asn-398) variant have lower function than those that incorporate α5 subunits with the common (Asp-398) variant (
      • Bierut L.J.
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      • Wang J.C.
      • Hinrichs A.L.
      • Grucza R.A.
      • Xuei X.
      • Saccone N.L.
      • Saccone S.F.
      • Bertelsen S.
      • Fox L.
      • Horton W.J.
      • Breslau N.
      • Budde J.
      • Cloninger C.R.
      • Dick D.M.
      • Foroud T.
      • Hatsukami D.
      • Hesselbrock V.
      • Johnson E.O.
      • Kramer J.
      • Kuperman S.
      • Madden P.A.
      • Mayo K.
      • Nurnberger Jr., J.
      • Pomerleau O.
      • Porjesz B.
      • Reyes O.
      • Schuckit M.
      • Swan G.
      • Tischfield J.A.
      • Edenberg H.J.
      • Rice J.P.
      • Goate A.M.
      Variants in nicotinic receptors and risk for nicotine dependence.
      ,
      • Kuryatov A.
      • Berrettini W.
      • Lindstrom J.
      Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function.
      ). This provides a mechanism through which the α5(D398N) mutation could produce phenotypic effects. Notably, a restricted set of brain regions (most prominently in the habenuolopeduncular pathway) express α5 subunits in combination with α3 and β4 subunits (
      • Zoli M.
      • Le Novère N.
      • Hill Jr., J.A.
      • Changeux J.P.
      Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems.
      ,
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      • Maskos U.
      • Ibañez-Tallon I.
      Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula.
      ), as often occurs in autonomic α3β4* nAChR (
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      • Simeone X.
      • Orr-Urtreger A.
      • Papke R.L.
      • McIntosh J.M.
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      Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes.
      ,
      • Conroy W.G.
      • Berg D.K.
      Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions.
      ,
      • Vernallis A.B.
      • Conroy W.G.
      • Berg D.K.
      Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes.
      ). A recent study showed that increased expression of α3β4* nAChR in the habenulopeduncular tract of mice increases nicotine aversion, an effect that can be reduced by the introduction and expression of additional α5(Asn-398) subunits in the same pathway (
      • Frahm S.
      • Slimak M.A.
      • Ferrarese L.
      • Santos-Torres J.
      • Antolin-Fontes B.
      • Auer S.
      • Filkin S.
      • Pons S.
      • Fontaine J.F.
      • Tsetlin V.
      • Maskos U.
      • Ibañez-Tallon I.
      Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula.
      ). Furthermore, α3, β4, and α5 nAChR subunits are commonly expressed in bronchial, epithelial, and lung cancer cells, where nAChR activation by nicotine has been proposed as a mechanism that may increase tumor initiation and/or growth (
      • Egleton R.D.
      • Brown K.C.
      • Dasgupta P.
      Nicotinic acetylcholine receptors in cancer. Multiple roles in proliferation and inhibition of apoptosis.
      ). However, heterologous expression studies done to date have not identified functional differences induced by α5 variant incorporation into α3β4* nAChR (
      • Kuryatov A.
      • Berrettini W.
      • Lindstrom J.
      Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function.
      ,
      • Li P.
      • McCollum M.
      • Bracamontes J.
      • Steinbach J.H.
      • Akk G.
      Functional characterization of the α5 (Asn-398) variant associated with risk for nicotine dependence in the α3β4α5 nicotinic receptor.
      ).
      Other observations may help to explain this discrepancy between in vitro observations and in vivo phenotypes. It has been shown that α3β4 nAChR can be expressed in multiple stoichiometries, with different functional properties (
      • Papke R.L.
      • Wecker L.
      • Stitzel J.A.
      Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
      ,
      • Krashia P.
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      • Sivilotti L.G.
      Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.
      ,
      • Grishin A.A.
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      • Alewood P.F.
      • Lewis R.J.
      • Adams D.J.
      α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors.
      ). Moreover, α5 subunits can “compete” with β4 subunits for incorporation into assembled nAChR (
      • Gahring L.C.
      • Rogers S.W.
      Nicotinic receptor subunit α5 modifies assembly, up-regulation, and response to pro-inflammatory cytokines.
      ), possibly forcing formation of non-functional nAChR subunit assemblies as “dead end intermediates” (
      • Kuryatov A.
      • Onksen J.
      • Lindstrom J.
      Roles of accessory subunits in α4β2(∗) nicotinic receptors.
      ). Thus, the effect(s) of common α5(Asp-398) versus risk α5(Asn-398) variant subunit incorporation into α3β4* nAChR may be obscured by changes, attendant on any α5 subunit incorporation, in the overall level of α3β4 nAChR functional expression and/or the balance of functional stoichiometric isoforms expressed. This complication in experimental interpretation is compounded when various mixtures of nAChR subtypes with specific subunit ratios are expressed from “loose” subunits assembled under host cell, and not investigator, control.
      To overcome these difficulties in interpretation, we employed a concatemeric nAChR approach (Fig. 1). Here, nAChR constructs are assembled that encode all five subunits of the desired α3β4* nAChR subtypes joined by short peptide linkers. The advantage of this approach is that complex nAChR subtypes can be expressed with native nAChR-like properties and with completely defined subunit ratios and orders of assembly (
      • Carbone A.L.
      • Moroni M.
      • Groot-Kormelink P.J.
      • Bermudez I.
      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ,
      • Kuryatov A.
      • Lindstrom J.
      Expression of functional human α6β2β3∗ acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.
      ). Using concatemeric α3β4α5 nAChR, we demonstrate that, as is true for α4β2* nAChR, incorporation of the α5(Asn-398) variant reduces maximal acetylcholine-induced function when compared with the α5(Asp-398) variant. The properties of the defined concatemeric nAChR also were compared with those of α3β4* nAChR allowed to assemble freely from loose individual subunits. These comparisons confirmed that concatemeric and freely assembled α3β4α5 nAChR have essentially indistinguishable pharmacological properties. Interestingly, these comparisons also suggested that loose α3 and β4 subunits associate quite differently in the presence or absence of α5 subunits.
      Figure thumbnail gr1
      FIGURE 1Linked (concatemeric) subunit design of α3β4α5 receptors. A, shown is a schematic illustration of (from top to bottom) β4α3β4α3β4, β4α3β4α3α3, β4α3β4α3α5(Asp-398), and β4α3β4α3α5(Asn-398) constructs. Each construct is flanked with AscI and EcoRV restriction sites (5′ and 3′, respectively; indicated by gray circles) for subcloning into high expression oocyte vectors. Kozac and the β4 signal peptide (SP) were retained only for the 1st position. Flanking each subunit position are unique restriction sites (indicated by gray circles) used in concatemer design (for example, AscI and XbaI used in exchanging nAChR subunits at position 1; XbaI and AgeI sites were used in exchanging nAChR subunits at position 2; etc.). Concatemers varied in composition only at position 5, containing either the β4, α3, or two naturally occurring variants of the α5 nAChR subunit (aspartic acid (Asp-398) or asparagine (Asn-398)). Stop codons (SC) were added at the 3′ end of subunit position 5. The number of AGS repeats flanking each subunit is listed below each linker region. B, shown is stoichiometry of β4α3β4α3β4, β4α3β4α3α3, β4α3β4α3α5(Asp-398), and β4α3β4α3α5(Asn-398) constructs. Concatemers form pentameric receptors by joining the positive interface of the nAChR subunit at position 1 (P1) and the negative interface of position 5 (P5). Restriction digest (below each schematic) using unique restriction sites (as mentioned above) was used to verify each subunit within its respective position (P1-P5). An additional restriction digest (*) using ScaI was performed to diagnose correct subunit composition and order. M, molecular mass markers.

      DISCUSSION

      The pentameric concatemer approach allows accurate and consistent reproduction of complex nAChR subtypes, with complete control over subunit ratios and associations (
      • Carbone A.L.
      • Moroni M.
      • Groot-Kormelink P.J.
      • Bermudez I.
      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ,
      • Kuryatov A.
      • Lindstrom J.
      Expression of functional human α6β2β3∗ acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.
      ,
      • Mazzaferro S.
      • Benallegue N.
      • Carbone A.
      • Gasparri F.
      • Vijayan R.
      • Biggin P.C.
      • Moroni M.
      • Bermudez I.
      Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2)2α4 nicotinic receptor influences agonist sensitivity.
      ,
      • Groot-Kormelink P.J.
      • Broadbent S.
      • Beato M.
      • Sivilotti L.G.
      Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs.
      ). It also allows for mutagenesis of a single subunit within an entire nAChR complex even where multiple copies of the target subunit may be present. These unique advantages were central to the work presented in this study. Using concatemeric α3β4α5 nAChR, we show that α5 subunit risk variant (Asn-398) incorporation reduces ACh-evoked function when compared with inclusion of the α5 common variant (Asp-398). Coexpression of unlinked α3, β4, and α5 subunits enforces assembly of an apparently uniform nAChR population with very similar pharmacological properties to those of concatemeric α3β4* nAChR. In addition, either variant of the α5 subunit is capable of reducing the overall amount of α3β4* nAChR function after coinjection with non-concatenated α3 and β4 subunits. Further observations suggested that removing the constraints imposed by either concatemerization or by co-expression with unlinked α5 subunits allows loose α3 and β4 subunits to assemble into at least two further subtypes. These α3β4-only subtypes have substantially different pharmacological profiles from each other, from unlinked subunit α3β4α5 nAChR, and from any of the concatenated α3β4 or α3β4α5 nAChR.
      Critically, the pharmacological properties of α3β4α5 nAChR expressed using pentameric concatemers were similar to those of the same subtype expressed from unlinked subunits. This finding indicates that the addition of the concatemeric linkers did not noticeably alter nAChR function. It also reinforces further that pentameric concatemers faithfully replicate the ligand sensitivity of the equivalent subunit arrangement when formed from loose subunits. It has been suggested that α5 subunits compete with β4 subunits (
      • Frahm S.
      • Slimak M.A.
      • Ferrarese L.
      • Santos-Torres J.
      • Antolin-Fontes B.
      • Auer S.
      • Filkin S.
      • Pons S.
      • Fontaine J.F.
      • Tsetlin V.
      • Maskos U.
      • Ibañez-Tallon I.
      Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula.
      ,
      • Gahring L.C.
      • Rogers S.W.
      Nicotinic receptor subunit α5 modifies assembly, up-regulation, and response to pro-inflammatory cytokines.
      ), reducing expression of functional α3β4α5 nAChR, possibly by encouraging the formation of dead-end intermediates that become trapped inside the cell (
      • Kuryatov A.
      • Onksen J.
      • Lindstrom J.
      Roles of accessory subunits in α4β2(∗) nicotinic receptors.
      ). Our observations support this concept. Coinjection of non-concatenated α5 subunit mRNA approximately halved α3β4* functional expression in Xenopus oocytes compared with injection of loose α3 and β4 subunits only (1:1:1 or 1:1 ratios were used; see the legend to Fig. 2). In contrast, if the α5 subunit is forced by concatemerization to assemble only as part of a pentameric nAChR complex, its incorporation substantially increases functional expression (Fig. 5). Together, these observations suggest that a reduction in function is not caused by the incorporation of α5 subunits per se. Instead, the presence of loose α5 subunits likely adversely affects the efficiency of unlinked α3 and β4 nAChR subunit assembly into functional nAChR.
      In contrast, α3β4-only nAChR expressed from pentameric concatemers had different pharmacological properties from those expressed from loose subunits (Table 1, top two rows, and Table 2). This discrepancy could be explained in several ways. One possibility is that covalent linkers may alter the properties of concatemeric nAChR by constraining structural transitions that are essential for normal function. This concern is mitigated by previous publications (
      • Carbone A.L.
      • Moroni M.
      • Groot-Kormelink P.J.
      • Bermudez I.
      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ,
      • Kuryatov A.
      • Lindstrom J.
      Expression of functional human α6β2β3∗ acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.
      ,
      • Mazzaferro S.
      • Benallegue N.
      • Carbone A.
      • Gasparri F.
      • Vijayan R.
      • Biggin P.C.
      • Moroni M.
      • Bermudez I.
      Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2)2α4 nicotinic receptor influences agonist sensitivity.
      ,
      • Groot-Kormelink P.J.
      • Broadbent S.
      • Beato M.
      • Sivilotti L.G.
      Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs.
      ) indicating that well designed pentameric nAChR concatemers can accurately reproduce the properties of multiple native nAChR subtypes (which assemble from unlinked subunits). In addition, the linkers in each of the pentameric concatemers used in this study are of the same length and composition; it is unlikely that only the non-α5* concatemers used in this study would suffer from linker-induced functional alterations. Furthermore, if the non-α5* concatemers were uniquely affected by the presence of the linkers, it would be expected that this would strongly alter agonist potencies and relative efficacies when compared with those of the α5* concatemers. This is not the case; the pharmacological parameters measured from all four of the concatemers tested here are strikingly similar. A second possibility is that the covalent linkers within the concatemers might break down. This would release sub-pentameric products that could assemble to form unintended, but functional, byproducts (
      • Zhou Y.
      • Nelson M.E.
      • Kuryatov A.
      • Choi C.
      • Cooper J.
      • Lindstrom J.
      Human α4β2 acetylcholine receptors formed from linked subunits.
      ,
      • Groot-Kormelink P.J.
      • Broadbent S.D.
      • Boorman J.P.
      • Sivilotti L.G.
      Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors.
      ,
      • Nicke A.
      • Rettinger J.
      • Schmalzing G.
      Monomeric and dimeric byproducts are the principal functional elements of higher order P2X1 concatamers.
      ). The presence of such degradation products was checked for by coinjection with an α5(V9′S) mutant subunit. Assembly of this mutant subunit with either single α3 and β4 subunits or subpentameric concatemers would result in a substantial gain of function (
      • Groot-Kormelink P.J.
      • Broadbent S.D.
      • Boorman J.P.
      • Sivilotti L.G.
      Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors.
      ,
      • Labarca C.
      • Nowak M.W.
      • Zhang H.
      • Tang L.
      • Deshpande P.
      • Lester H.A.
      Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors.
      ). No change in function was noted when α5(V9′S) was co-injected with a concatemeric construct. This confirms that all, or nearly all, of the function in oocytes injected with pentameric nAChR mRNA constructs arises from fully-pentameric concatemeric nAChR. Finally, and most likely, the precise subunit associations imposed by concatemeric constructs may, or may not, correspond to those favored during association of loose subunits. Our data suggest that the α3β4α5 concatemers accurately reproduce the conformation adopted when the relevant individual subunits assemble freely. However, the same is not true for the α3β4-only constructs when compared with nAChR assembled from loose α3 and β4 subunits. This would indicate that one role of the α5 subunit is to impose a particular subunit composition on α3β4* nAChR expressed from loose subunits. If α5 is a true “accessory” subunit (i.e. does not interact directly with ligands), this may be unavoidable; a (α3β4)2α5 conformation is the only one in which two pairs of α3+β4 subunits would be available to provide agonist binding pockets and thus to assemble a functional α3β4α5 nAChR.
      The preceding observations raise the question of which nAChR subtype(s) is expressed after coinjection of only α3 and β4 subunits. This study confirms prior reports that at least two α3β4 nAChR populations may be formed and that their relative expression levels depend on the molar injection ratio of the subunit mRNAs (1:20 versus 20:1). The pharmacology observed in this study matches that reported in other recent publications (
      • Krashia P.
      • Moroni M.
      • Broadbent S.
      • Hofmann G.
      • Kracun S.
      • Beato M.
      • Groot-Kormelink P.J.
      • Sivilotti L.G.
      Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.
      ,
      • Grishin A.A.
      • Wang C.I.
      • Muttenthaler M.
      • Alewood P.F.
      • Lewis R.J.
      • Adams D.J.
      α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors.
      ) that used less-extreme injection ratios (1:9 versus 9:1 or 1:10 versus 10:1). The lack of further changes in observed pharmacology after adoption of more extreme subunit ratios indicates that, as for α4 and β2 subunits (
      • Zwart R.
      • Vijverberg H.P.
      Four pharmacologically distinct subtypes of α4β2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.
      ,
      • Moroni M.
      • Vijayan R.
      • Carbone A.
      • Zwart R.
      • Biggin P.C.
      • Bermudez I.
      Non-agonist binding subunit interfaces confer distinct functional signatures to the alternate stoichiometries of the α4β2 nicotinic receptor. An α4-α4 interface is required for Zn2+ potentiation.
      ,
      • Harps⊘e K.
      • Ahring P.K.
      • Christensen J.K.
      • Jensen M.L.
      • Peters D.
      • Balle T.
      Unraveling the high and low sensitivity agonist responses of nicotinic acetylcholine receptors.
      ), relatively pure populations of two different α3β4 subunit assemblies are produced at the injection ratios used in this study. The same studies proposed again by analogy to the well-studied α4β2 nAChR that the different nAChR isoforms might correspond to (α3β4)2β4 and (α3β4)2α3 nAChR (
      • Krashia P.
      • Moroni M.
      • Broadbent S.
      • Hofmann G.
      • Kracun S.
      • Beato M.
      • Groot-Kormelink P.J.
      • Sivilotti L.G.
      Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.
      ).
      Accordingly we constructed (α3β4)2β4 and (α3β4)2α3 concatemers using the same subunit arrangements as used successfully to encode high and low agonist sensitivity pentameric α4β2 nAChR concatemers (
      • Carbone A.L.
      • Moroni M.
      • Groot-Kormelink P.J.
      • Bermudez I.
      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ). We initially anticipated that these concatemers would have similar pharmacological profiles to α3β4-only nAChR formed after injection of loose α3 and β4 subunits at 1:20 and 20:1 ratios, respectively. However, the pharmacology observed after injection of loose α3 and β4 subunits at either 1:20 or 20:1 ratios (Fig. 6, Table 2) was strikingly different from the concatemeric “(α3β4)2X-type” measurements. The precise arrangements adopted by loose α3 and β4 subunits injected at different ratios remain unknown. It certainly seems probable that 1:20 and 20:1 α3:β4 injection ratios may give rise to nAChR with different stoichiometries (
      • Krashia P.
      • Moroni M.
      • Broadbent S.
      • Hofmann G.
      • Kracun S.
      • Beato M.
      • Groot-Kormelink P.J.
      • Sivilotti L.G.
      Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.
      ,
      • Grishin A.A.
      • Wang C.I.
      • Muttenthaler M.
      • Alewood P.F.
      • Lewis R.J.
      • Adams D.J.
      α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors.
      ). In addition, as demonstrated for GABAA receptors, the precise order of subunit incorporation (even for identical subunit stoichiometries) can affect receptor function (
      • Sigel E.
      • Baur R.
      • Boulineau N.
      • Minier F.
      Impact of subunit positioning on GABAA receptor function.
      ). The emerging awareness that agonist binding to non-canonical nAChR interfaces can strongly affect function underlines this point (
      • Carbone A.L.
      • Moroni M.
      • Groot-Kormelink P.J.
      • Bermudez I.
      Pentameric concatenated (α4)(2)(β2)(3) and (α4)(3)β2)(2) nicotinic acetylcholine receptors. Subunit arrangement determines functional expression.
      ,
      • Harps⊘e K.
      • Ahring P.K.
      • Christensen J.K.
      • Jensen M.L.
      • Peters D.
      • Balle T.
      Unraveling the high and low sensitivity agonist responses of nicotinic acetylcholine receptors.
      ,
      • Seo S.
      • Henry J.T.
      • Lewis A.H.
      • Wang N.
      • Levandoski M.M.
      The positive allosteric modulator morantel binds at noncanonical subunit interfaces of neuronal nicotinic acetylcholine receptors.
      ). Determining whether different α3:β4 subunit mRNA injection ratios produce nAChR with different stoichiometries, different arrangements of the same subunit stoichiometries, or both will require a great deal more investigation. The concatemeric pentamer approach is uniquely well suited to addressing this question.
      Unlike agonist EC50 values, IC50 values for mecamylamine inhibition were greatly affected by the identity of the fifth subunit in each pentameric concatemer. This suggests that mecamylamine (a non-competitive antagonist) interacts with the resulting nAChR in a position where it can be influenced by the presence of alternate subunits in the fifth, non-agonist-binding position. This sensitivity to α3β4* nAChR composition was also evident when comparing mecamylamine IC50 values between α3β4 and α3β4α5 nAChR expressed from loose subunits (Table 1). These observations indicate that non-competitive ligands may provide the best opportunities to pharmacologically distinguish between different subunit arrangements of α3β4* isoforms. Importantly, this category could also include positive allosteric modulators and/or allosteric agonists in addition to non-competitive antagonists. Given the association of α5 subunit variants with a variety of substance abuse behaviors (see introduction), selective manipulation of α3β4α5 nAChR activity could have valuable therapeutic implications.
      Functional effects of the α5(D398N) mutation are hard to distinguish without using a fully pentameric concatemer approach. The previously described effects of α5 subunits on the efficiency of α3β4* nAChR expression and possibly also on subunit associations/assembly could outweigh and obscure the effects of the α5(D398N) mutation. This could explain previous studies' conclusions that the effects of α5(Asp-389) incorporation were the same as those of α5(Asn-389) (
      • Kuryatov A.
      • Berrettini W.
      • Lindstrom J.
      Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function.
      ,
      • Li P.
      • McCollum M.
      • Bracamontes J.
      • Steinbach J.H.
      • Akk G.
      Functional characterization of the α5 (Asn-398) variant associated with risk for nicotine dependence in the α3β4α5 nicotinic receptor.
      ,

      Stokes, C., Papke, R. L. (2012) Neuropharmacology, in press

      ). However, using a pentameric concatemer approach, we were able to compare the function of uniform populations of (α3β4)2α5(Asp-389) versus (α3β4)2α5(Asn-389) nAChR. The maximum ACh-induced function produced by (α3β4)2α5(Asp-389) nAChR was significantly greater than that measured for (α3β4)2α5(Asn389) nAChR (Fig. 5). Increased function for α5(Asp-398)* versus α5(Asn-398)* nAChR with little difference in pharmacological profile matches previous observations regarding α5 variant incorporation into α4β2* nAChR (
      • Bierut L.J.
      • Stitzel J.A.
      • Wang J.C.
      • Hinrichs A.L.
      • Grucza R.A.
      • Xuei X.
      • Saccone N.L.
      • Saccone S.F.
      • Bertelsen S.
      • Fox L.
      • Horton W.J.
      • Breslau N.
      • Budde J.
      • Cloninger C.R.
      • Dick D.M.
      • Foroud T.
      • Hatsukami D.
      • Hesselbrock V.
      • Johnson E.O.
      • Kramer J.
      • Kuperman S.
      • Madden P.A.
      • Mayo K.
      • Nurnberger Jr., J.
      • Pomerleau O.
      • Porjesz B.
      • Reyes O.
      • Schuckit M.
      • Swan G.
      • Tischfield J.A.
      • Edenberg H.J.
      • Rice J.P.
      • Goate A.M.
      Variants in nicotinic receptors and risk for nicotine dependence.
      ,
      • Kuryatov A.
      • Berrettini W.
      • Lindstrom J.
      Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function.
      ).
      It appears that, as previously proposed (
      • Frahm S.
      • Slimak M.A.
      • Ferrarese L.
      • Santos-Torres J.
      • Antolin-Fontes B.
      • Auer S.
      • Filkin S.
      • Pons S.
      • Fontaine J.F.
      • Tsetlin V.
      • Maskos U.
      • Ibañez-Tallon I.
      Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula.
      ), α5 subunit expression may act to modulate the amount of α3β4* nAChR function in the habenulopeduncular tract and in other tissues that express α3β4α5 nAChR. This study indicates that the presence of the α5(Asp-398) or α5(Asn-398) variant will impose an additional layer of functional modulation. As noted previously (
      • Kuryatov A.
      • Berrettini W.
      • Lindstrom J.
      Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function.
      ), the concentrations of nicotine present in smokers are too low to significantly activate or desensitize α3β4α5 nAChR. However, the activity induced by synaptic or perisynaptic ACh release onto α3β4* nAChR could be strongly affected by the integration of α5(Asp-398) or α5(Asn-398) subunits. This in turn could result in compensatory changes either at the neurotransmitter/receptor level or at the circuit activity level, which may explain some of the phenotypic variations attributed to the α5(D398N) mutation. Given the established role of the habenulopeduncular pathway α3β4α5 nAChR function in nicotine dependence and aversive behavior (
      • Frahm S.
      • Slimak M.A.
      • Ferrarese L.
      • Santos-Torres J.
      • Antolin-Fontes B.
      • Auer S.
      • Filkin S.
      • Pons S.
      • Fontaine J.F.
      • Tsetlin V.
      • Maskos U.
      • Ibañez-Tallon I.
      Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula.
      ,
      • Fowler C.D.
      • Lu Q.
      • Johnson P.M.
      • Marks M.J.
      • Kenny P.J.
      Habenular α5 nicotinic receptor subunit signaling controls nicotine intake.
      ,
      • Salas R.
      • Orr-Urtreger A.
      • Broide R.S.
      • Beaudet A.
      • Paylor R.
      • De Biasi M.
      The nicotinic acetylcholine receptor subunit α 5 mediates short term effects of nicotine in vivo.
      ), it seems likely that selective manipulation of α3β4α5 function mediated by this subtype could represent a valuable smoking cessation strategy. Our current findings indicate that non-competitive/allosteric compounds may be the most promising category of potential therapeutic agents for such an approach.

      Acknowledgments

      We thank Minoti Bhakta for technical assistance with concatemer design and construction.

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