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Modulation of Gain-of-function α6*-Nicotinic Acetylcholine Receptor by β3 Subunits*

  • Bhagirathi Dash
    Footnotes
    Affiliations
    Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013
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  • Ronald J. Lukas
    Correspondence
    To whom correspondence should be addressed: Division of Neurobiology, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix, AZ 85013. Tel.: 602-406-3399; Fax: 602-406-4172;
    Affiliations
    Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant DA015389 (to R. J. L.), by Philip Morris USA Inc. and Philip Morris International through their External Research Program, and by endowment and capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation. Portions of this work have been presented in abstract form (Dash, B., Chang, Y., and Lukas, R. J. (2008) Molecular and pharmacological characterization of recombinant α6β4*-nAChR. Soc. Neurosci. Abst. 34, 233.13).
    1 Present address: Dept. of Psychiatry and Neurobehavioral Sciences, School of Medicine, University of Virginia, Charlottesville, VA 22911.
Open AccessPublished:February 07, 2012DOI:https://doi.org/10.1074/jbc.M111.322610
      We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6(L9′S or V13′S)β4*- or α6(N143D+M145V)L9′Sβ2*-nAChR. This contrasts with the lack of consistent function when α6(L9′S or V13′S) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα6(N143D+M145V)L9′Shβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9′ or 13′ mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR.

      Introduction

      Nicotinic acetylcholine receptors (nAChR)
      The abbreviations used are: ACh
      acetylcholine
      nAChR
      nicotinic acetylcholine receptor(s)
      Imax
      peak current response
      m
      mouse
      h
      human.
      are pentameric ligand-gated ion channels expressed throughout the nervous system. Those other than the muscle-type (embryonic α1β1γδ- or adult α1β1γϵ-) nAChR are thought to be composed of different permutations of eight α subunits (α2-α7, α9-α10) and three β subunits (β2-β4) in humans (
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      International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits.
      ). Of specific interest to us in this study are α6β3*-nAChR (where * indicates the known or possible presence of nAChR subunits other than those specified) (
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      Functional nicotinic acetylcholine receptors containing α6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons.
      ,
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ). α6β3*-nAChR have been implicated in dopaminergic neurotransmission, nicotine dependence, anxiety, and other important neurophysiological processes (
      • Cui C.
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      ,
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      α6-Containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens.
      ,
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      The neuronal nicotinic acetylcholine receptors α4* and α6* differentially modulate dopamine release in mouse striatal slices.
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      Crucial role of α4 and α6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration.
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      Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs.
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      Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area α6β2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement.
      ,
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      Decreased anxiety-like behavior in β3 nicotinic receptor subunit knockout mice.
      ,
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      Cholinergic modulation of locomotion and striatal dopamine release is mediated by α6α4* nicotinic acetylcholine receptors.
      ,
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      • Heintz N.
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      • Bencherif M.
      • Marks M.J.
      • Lester H.A.
      In vivo activation of midbrain dopamine neurons via sensitized, high affinity α6 nicotinic acetylcholine receptors.
      ).
      In vitro expression of functional, all-mouse (m) or all-human (h), wild-type α6β3*-nAChR has been difficult to achieve despite strong evidence for expression of α6β3*-nAChR in rodent brain (
      • Yang K.
      • Buhlman L.
      • Khan G.M.
      • Nichols R.A.
      • Jin G.
      • McIntosh J.M.
      • Whiteaker P.
      • Lukas R.J.
      • Wu J.
      Functional nicotinic acetylcholine receptors containing α6 subunits are on GABAergic neuronal boutons adherent to ventral tegmental area dopamine neurons.
      ,
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Exley R.
      • Clements M.A.
      • Hartung H.
      • McIntosh J.M.
      • Cragg S.J.
      α6-Containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens.
      ,
      • Meyer E.L.
      • Yoshikami D.
      • McIntosh J.M.
      The neuronal nicotinic acetylcholine receptors α4* and α6* differentially modulate dopamine release in mouse striatal slices.
      ,
      • Gotti C.
      • Guiducci S.
      • Tedesco V.
      • Corbioli S.
      • Zanetti L.
      • Moretti M.
      • Zanardi A.
      • Rimondini R.
      • Mugnaini M.
      • Clementi F.
      • Chiamulera C.
      • Zoli M.
      Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area α6β2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement.
      ,
      • Drenan R.M.
      • Grady S.R.
      • Steele A.D.
      • McKinney S.
      • Patzlaff N.E.
      • McIntosh J.M.
      • Marks M.J.
      • Miwa J.M.
      • Lester H.A.
      Cholinergic modulation of locomotion and striatal dopamine release is mediated by α6α4* nicotinic acetylcholine receptors.
      ,
      • Drenan R.M.
      • Grady S.R.
      • Whiteaker P.
      • McClure-Begley T.
      • McKinney S.
      • Miwa J.M.
      • Bupp S.
      • Heintz N.
      • McIntosh J.M.
      • Bencherif M.
      • Marks M.J.
      • Lester H.A.
      In vivo activation of midbrain dopamine neurons via sensitized, high affinity α6 nicotinic acetylcholine receptors.
      ,
      • Gotti C.
      • Moretti M.
      • Clementi F.
      • Riganti L.
      • McIntosh J.M.
      • Collins A.C.
      • Marks M.J.
      • Whiteaker P.
      Expression of nigrostriatal α6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by β3 subunit gene deletion.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ). Functional expression of α6*-nAChR only has been achieved in Xenopus oocytes when using specific forms of mutant or chimeric subunits or in hybrid α6*-nAChR composed of subunits from different species (
      • Fucile S.
      • Matter J.M.
      • Erkman L.
      • Ragozzino D.
      • Barabino B.
      • Grassi F.
      • Alemà S.
      • Ballivet M.
      • Eusebi F.
      The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells.
      ,
      • Evans N.M.
      • Bose S.
      • Benedetti G.
      • Zwart R.
      • Pearson K.H.
      • McPhie G.I.
      • Craig P.J.
      • Benton J.P.
      • Volsen S.G.
      • Sher E.
      • Broad L.M.
      Expression and functional characterization of a human chimeric nicotinic receptor with α6β4 properties.
      ,
      • Gerzanich V.
      • Kuryatov A.
      • Anand R.
      • Lindstrom J.
      “Orphan” α6 nicotinic AChR subunit can form a functional heteromeric acetylcholine receptor.
      ,
      • Kuryatov A.
      • Olale F.
      • Cooper J.
      • Choi C.
      • Lindstrom J.
      Human α6 AChR subtypes: subunit composition, assembly, and pharmacological responses.
      ,
      • Kuryatov A.
      • Lindstrom J.
      Expression of functional human α6β2β3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.
      ). For example, function is achieved when chimeric, hα6/hα3 subunits (composed of the N-terminal, first extracellular domain of the hα6 subunit fused to the first transmembrane domain through to the C terminus of the hα3 subunit) are coexpressed with hβ2 or hβ4 subunits alone or in the presence of hβ3 subunits (
      • Kuryatov A.
      • Olale F.
      • Cooper J.
      • Choi C.
      • Lindstrom J.
      Human α6 AChR subtypes: subunit composition, assembly, and pharmacological responses.
      ). α6*-nAChR are functional when expressed as hybrids of mouse and human α6 and other subunits, and there is function of some complexes containing β3 subunits mutated at specific residues in their second transmembrane domains (leucine- or valine-to-serine mutations at 9′ or 13′ positions; L9′S or V13′S) to confer gain-of-function effects (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ,
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ). Potentiation of function is sometimes seen when wild-type β3 subunits are incorporated into hybrid complexes, but this is in contrast to dominant-negative effects of coexpression with wild-type β3 subunits on function of α6β4*-nAChR when all subunits are from the same species (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ). There may be host cell specificity in some of these effects because nAChR hβ3 subunits promote expression and nicotine-induced up-regulation of h6*-nAChR in transfected cell lines (
      • Tumkosit P.
      • Kuryatov A.
      • Luo J.
      • Lindstrom J.
      β3 subunits promote expression and nicotine-induced up-regulation of human nicotinic α6* nicotinic acetylcholine receptors expressed in transfected cell lines.
      ).
      We and others have taken advantage of gain-of-function mutations in the nAChR β3 subunit to produce functional nAChR, including those containing α6 subunits, in part to assess capabilities of subunits to coassemble, but also as a strategy to increase functional gain (signal:noise) to facilitate such assessments (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ,
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ). For example, coexpression with β3V9′S subunits increases agonist sensitivity and efficacy for α6*-nAChR. We hypothesized that similar mutations in nAChR α6 subunits would increase agonist sensitivity and efficacy of α6(L9′S or V13′S)(β4 or β2)*-nAChR to provide enough functional gain to facilitate evaluation of effects of wild-type β3 subunits on complexes and even to ensure that we can detect incorporation of wild-type β3 subunits into α6(L9′S or V13′S)*-nAChR. We also hypothesized that wild-type β3 subunits would have the same effects, dominant-negative or potentiating, depending on the subunit combination investigated, on gain-of-function α6(L9′S or V13′S)*-nAChR as they did on wild-type α6*-nAChR. This would help us assess whether any reduction or abolishment of function is due to altered open channel probability (
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ) or due to reduced surface expression of nAChR because β3 subunit incorporation facilitates formation of dead end intermediates (
      • Kuryatov A.
      • Onksen J.
      • Lindstrom J.
      Roles of accessory subunits in α4β2* nicotinic receptors.
      ). Our results indicated that whenever nAChR β3 subunits are incorporated into (α6 or hα6(N143D+M145V))(L9′S or V13′S)*-nAChR, function is potentiated (i.e. there is higher agonist potency and larger magnitude responses) irrespective of whether there are dominant-negative or potentiating effects of β3 subunits on wild-type α6*-nAChR.

      DISCUSSION

      Recent studies have investigated how nAChR β3 subunits might incorporate as accessory partners into nAChR subtypes, specifically into α6*-nAChR (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ). To further understand how β3 subunits might incorporate into α6*-nAChR, we exploited the gain-of-function/reporter mutant strategy (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ,
      • 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.
      ) to reveal whether β3 subunits integrate into α6*-nAChR complexes that are on the cell surface and functional. This approach allows us to focus on cell surface, functional receptors without complications due to ambiguities of protein chemical or immunochemical studies confounded by the prevalent expression of intracellular and perhaps partially assembled receptor complexes and the unreliable quality and/or availability of most anti-nAChR antibodies for use in immunoprecipitation and/or immunoblot studies (
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ). In addition, we based the current studies on our findings (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ) that (i) incorporation of nAChR β3 subunits into α6*-nAChR, mouse or human, has a dominant-negative effect; (ii) incorporation of nAChR hβ3 subunits into mα6hβ4*- or mα6mβ4*- nAChR leads to formation of functional nAChR; and (iii) mutations in the E1 N-terminal domain of the nAChR hα6 subunit are essential for successful assembly and formation of functional hα6(N143D+M145V)hβ2hβ3V9′S-nAChR.
      The principal findings of this study, whenever functional expression levels are adequate to allow comparisons, and with exceptions that could be informative as discussed below, are: (i) that introduction of 9′ or 13′ mutations into the second transmembrane domain of mα6 or hα6 subunits typically has a gain-of-function effect, leading to production of (α6 or α6(N143D+M145V))(L9′S or V13′S)(β2 or β4)*-nAChR that have 6–34-fold higher sensitivity to nicotine and much higher levels of function than do nAChR containing the same subunit combinations but with wild-type α6 subunits; (ii) that incorporation of β3 subunits into (α6 or α6(N143D+M145V))(L9′S or V13′S)(β2 or β4)*-nAChR typically increases levels of receptor function with or without concomitant increase in agonist potency; and (iii) that gain-of-function mutations in α6 or α6(N143D+M145V) subunits still do not allow for formation of functional α6(L9′S or V13′S)β2-nAChR complexes, thus continuing to confound assessments of roles played by β3 subunits in modulation of α6β2*-nAChR.
      The amount of functional expression for hα6L9′Shβ4-, mα6L9′Smβ4-, or mα6L9′Shβ4-nAChR is modest in absolute terms (27–80-nA peak current). However, with the exception of the insignificant difference in the magnitude of function seen for all-mouse mα6L9′Smβ4- and mα6mβ4-nAChR, the increase in function upon expression with the α6 subunit 9′ mutants is remarkable because of the lack of reliable function for wild-type, all human hα6hβ4-, or hybrid mα6hβ4-nAChR. The little-if-any function for all-wild-type α6β4-nAChR complicates quantitative assessment of effects of α6 subunit gain-of-function mutations on agonist potency, although qualitatively, nicotine EC50 values are over 10 μm for α6β4-nAChR and never higher than 3.1 μm for α6(L9′S or V13′S)β4-nAChR. However, gain-of-function effects manifest as increases in agonist potency and in peak current magnitudes are very clear based on comparisons of hα6hβ4hβ3- with hα6L9′Shβ4hβ3-nAChR and comparisons of mα6hβ4hβ3- with mα6L9′Shβ4hβ3-nAChR. A difference in agonist potency is also clear for comparison of mα6mβ4hβ3- with mα6L9′Smβ4hβ3-nAChR, although there is only a 2-fold difference in peak current response across these receptors, partly due to the relatively high absolute levels of function for the hybrid mα6mβ4hβ3-nAChR. Once again, however, all-mouse α6β4β3-nAChR are outliers because there is only modest function for mα6L9′Smβ4mβ3-nAChR, although there is no reliable function for the all-wild-type analog, mα6mβ4mβ3-nAChR.
      Nevertheless, and very interestingly, for all-mouse α6*-receptors, although there is not reproducible function for mα6V13′Smβ4-nAChR, there are increases both in agonist potency and in response magnitude for mα6V13′Smβ4mβ3-nAChR when compared with those parameters for any form of mα6mβ4-nAChR or for mα6mβ4mβ3- or mα6L9′Smβ4mβ3-nAChR. Our initial studies of mouse α6*-nAChR were prompted because of the reported difficulties in heterologous expression of all-human α6*-nAChR and because so many data on naturally expressed α6*-nAChR function came from studies using rodents, but we have found all-mouse α6*-nAChR no easier to express than human α6*-nAChR. Expression of hybrid nAChR made up of subunits from different species has been more productive, suggesting that subtle differences for a given subunit across species in amino acid sequences in N-terminal, extracellular domains, but also in cytoplasmic and perhaps transmembrane domains, and at what must be at subunit interfaces not heretofore recognized as being functionally important, can strongly influence whether functional α6*-nAChR can be produced (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ). The fact that mα6 L9′S and V13′S mutations differing in position by just one turn in the second transmembrane domain α-helix can have such a large difference in their impact on mα6mβ4*-nAChR function indicates unexpectedly important roles for this channel-lining region in α6*-nAChR function. More work is warranted to more thoroughly characterize the bases for these influences.
      Our findings demonstrate that α6 subunit L9′S or V13′S modifications can function as reporter and/or gain-of-function mutations, leading to production of receptors with heightened sensitivity to agonists, thus confirming the presence of α6 subunits in functional receptor complexes, as expected. These studies also further affirm and recapture the strategy applied to exploit gain-of-function α6 subunit mutations expressed in vivo to enhance sensitivity to agonists and thus to help reveal roles played by α6*-nAChR in dopaminergic pathways relevant to movement disorders and nicotine dependence (
      • Drenan R.M.
      • Grady S.R.
      • Whiteaker P.
      • McClure-Begley T.
      • McKinney S.
      • Miwa J.M.
      • Bupp S.
      • Heintz N.
      • McIntosh J.M.
      • Bencherif M.
      • Marks M.J.
      • Lester H.A.
      In vivo activation of midbrain dopamine neurons via sensitized, high affinity α6 nicotinic acetylcholine receptors.
      ).
      This study was also initiated largely to assess whether effects previously described of nAChR β3 subunit incorporation into α6*-nAChR would be preserved when receptor functional levels at baseline were intentionally elevated by using reporter mutation/gain-of-function α6 subunits as coexpression partners. By contrast to earlier work by others (
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ), in which β3 subunits were coexpressed in excess over other subunits, we chose to introduce equal amounts of subunit cRNAs into oocytes for the current work, anticipating that approximately equal amounts of subunit proteins would be made and that this more closely approximates conditions in vivo. We confirmed our previous observations (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ) that hβ3 subunit incorporation into hα6hβ4*-nAChR has an uncertain effect on functional expression, that mβ3 subunit incorporation into mα6mβ4*-nAChR has a dominant-negative effect on receptor function, that hβ3 subunit incorporation into hybrid mα6hβ4*-nAChR potentiates function, but that there is even larger potentiation of function when hβ3 subunits are incorporated into hybrid mα6mβ4*-nAChR. However, with the exception of the lack of an obvious effect of mβ3 subunit incorporation on low functioning mα6L9′Smβ4*-nAChR, wild-type β3 subunit incorporation into any of the tested α6(L9′S or V13′S)β4-nAChR potentiated levels of function by >11-fold, notably including effects of hβ3 subunits on low functioning mα6L9′Smβ4*-nAChR and effects of mβ3 subunits on mα6V13′Smβ4*-nAChR. These findings indicate that β3 subunits do not always have dominant-negative effects on α6*-nAChR function as suggested earlier (
      • Broadbent S.
      • Groot-Kormelink P.J.
      • Krashia P.A.
      • Harkness P.C.
      • Millar N.S.
      • Beato M.
      • Sivilotti L.G.
      Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
      ) and do not always promote formation of dead end, α6β4*-nAChR intermediates as suggested previously (
      • Kuryatov A.
      • Onksen J.
      • Lindstrom J.
      Roles of accessory subunits in α4β2* nicotinic receptors.
      ). Instead, based on our results shown here, we can hypothesize that β3 subunits seem to promote assembly, cell surface expression, and/or functional responsiveness of α6β4*-nAChR, at least when there is enough function for α6β4(non-β3)-nAChR to allow assessment of effects of β3 subunit incorporation. Our findings using the oocyte expression system are in line with observations made regarding β3 subunit effects on α6*-nAChR functional expression in cell lines (
      • Tumkosit P.
      • Kuryatov A.
      • Luo J.
      • Lindstrom J.
      β3 subunits promote expression and nicotine-induced up-regulation of human nicotinic α6* nicotinic acetylcholine receptors expressed in transfected cell lines.
      ), suggesting that successful, functional α6β4*-nAChR expression in oocytes does not require coexpression with chaperones missing from oocytes but present in neurons or selected cell lines. Notably, although peak current potentiation upon substitution of gain-of-function α6 subunits (or β3 subunits; see Refs.
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      and
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ) occurs along with an increase in agonist potency, wild-type β3 incorporation into complexes increases peak current responses without affecting agonist potency.
      In almost every case, α6(L9′S or V13′S)*-nAChR spend a finite amount of time in a spontaneously open channel state, as judged by the ability of mecamylamine to block those open channels, giving the appearance of production of outward currents. This is a common feature for nAChR containing subunits with second transmembrane domain mutations that give gain-of-function effects (
      • Miko A.
      • Werby E.
      • Sun H.
      • Healey J.
      • Zhang L.
      A TM2 residue in the β1 subunit determines spontaneous opening of homomeric and heteromeric γ-aminobutyric acid-gated ion channels.
      ,
      • Chang Y.
      • Weiss D.S.
      Substitutions of the highly conserved M2 leucine create spontaneously opening rho1 γ-aminobutyric acid receptors.
      ). Interestingly, the absolute magnitudes of responses to mecamylamine generally are quite similar across all the α6*-nAChR studied (7.8–12 nA), even when magnitudes of agonist-induced inward currents varied much more widely (26–800 nA). The only exceptions are for mα6V13′Smβ4-nAChR, which curiously have no reproducible responses to nicotine or to mecamylamine, despite there being strong responses upon incorporation of mβ3 subunits to form mα6V13′Smβ4mβ3-nAChR, and for mα6L9′Shβ4hβ3-nAChR, which have slightly larger responses to mecamylamine (41 nA) but also have the largest responses to nicotine (870 nA).
      Although the current findings support a role for β3 subunits in potentiating function of α6β4*-nAChR with at least a modicum of baseline functional activity, we were confounded in our studies of α6β2*-nAChR by a general lack of function. This made it impossible to assess effects of β3 subunit incorporation on α6β2*-nAChR, but the results indicate that any gain-of-function earned by incorporation of α6(L9′S or V13′S) subunits into complexes is inadequate to reveal effects of β3 subunits, perhaps due to the surprising incompatibilities (illuminated in Refs.
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      and
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ) that often occur in attempts to use α6, β2, and β3 subunits to form functional receptors. In order for us to show that in fact a variant of gain-of-function hα6 subunit can partner with hβ2 and hβ3 subunit to form functional nAChR, we took advantage of our site-directed mutagenesis work (
      • Dash B.
      • Bhakta M.
      • Chang Y.
      • Lukas R.J.
      Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR.
      ,
      • Dash B.
      • Chang Y.
      • Lukas R.J.
      Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 subunits and/or variants modulate function of α6*-nAChR.
      ), which has implicated α6 residues 143 and 145 in the ability of β3 subunits to affect α6β2*-nAChR function. The hα6(N143D+M145V) mutations change the indicated residues to those that are in the mα6 subunit and permit mutated hα6 subunits to show function when coexpressed with hβ2 and hβ3 subunits when wild-type hα6 subunits do not. Human nAChR α6 subunit residues 143 and 145 are in the E1 domain, in loop E, on the (−) or complementary face of the subunit. This suggests that interactions between the α6 subunit (−) face with the (+) face from either β2 subunits or β3 subunits are important for functional α6*-nAChR expression. In order for us to prove that the nAChR β3 subunit does affect the function of α6β2*-nAChR, a 9′ mutation was introduced into the hα6(N143D+M145V) subunit. Although coexpression of hα6(N143D+M145V)L9′S and hβ2 subunits did not yield receptors with reliable function, upon inclusion of the hβ3 subunit, function was evident in all oocytes coexpressing the three subunits together. These hα6(N143D+M145V)L9′Shβ2*-nAChR mimic the gain-of-function, high-affinity mα6*-nAChR artificially expressed in mouse midbrain dopamine neurons (
      • Drenan R.M.
      • Grady S.R.
      • Whiteaker P.
      • McClure-Begley T.
      • McKinney S.
      • Miwa J.M.
      • Bupp S.
      • Heintz N.
      • McIntosh J.M.
      • Bencherif M.
      • Marks M.J.
      • Lester H.A.
      In vivo activation of midbrain dopamine neurons via sensitized, high affinity α6 nicotinic acetylcholine receptors.
      ).
      We conclude, based on the current and previous findings, that gain-of-function/reporter mutations introduced into α6 subunits in α6(β2 or β4)β3-nAChR are effective in potentiating receptor function. This potentiation yields receptors with higher agonist potency and larger magnitude responses to agonists, and also a finite likelihood of existing in a spontaneously open channel state. We also conclude from the present studies that wild-type β3 subunit incorporation into functionally competent (α6 or α6(N143D+M145V)) (L9′S or V13′S)(β4 or β2)*-nAChR has a potentiating effect irrespective of whether there are dominant-negative, null, or potentiating effects of β3 subunits on wild-type α6(β2 or β4)*-nAChR. In fact, reliable expression of functional gain-of-function α6*-nAChR is achieved only in the presence of nAChR β3 subunits. These results suggest that wild-type β3 subunit coexpression is at least permissive for cell surface expression of α6β4*-nAChR and very likely promotes function of these receptors. The strategies and results demonstrated here to increase function of α6*-nAChR to levels compatible with drug screening could facilitate the development of new drugs selective for α6*-nAChR. This is of increasing importance given the potentially important roles for α6*-nAChR in movement and movement disorders, mood disorders, and drug reinforcement (
      • Cui C.
      • Booker T.K.
      • Allen R.S.
      • Grady S.R.
      • Whiteaker P.
      • Marks M.J.
      • Salminen O.
      • Tritto T.
      • Butt C.M.
      • Allen W.R.
      • Stitzel J.A.
      • McIntosh J.M.
      • Boulter J.
      • Collins A.C.
      • Heinemann S.F.
      The β3 nicotinic receptor subunit: a component of α-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors.
      ,
      • Drenan R.M.
      • Grady S.R.
      • Whiteaker P.
      • McClure-Begley T.
      • McKinney S.
      • Miwa J.M.
      • Bupp S.
      • Heintz N.
      • McIntosh J.M.
      • Bencherif M.
      • Marks M.J.
      • Lester H.A.
      In vivo activation of midbrain dopamine neurons via sensitized, high affinity α6 nicotinic acetylcholine receptors.
      ,
      • Wang N.
      • Orr-Urtreger A.
      • Chapman J.
      • Rabinowitz R.
      • Nachman R.
      • Korczyn A.D.
      Autonomic function in mice lacking α5 neuronal nicotinic acetylcholine receptor subunit.
      ,
      • Salas R.
      • Orr-Urtreger A.
      • Broide R.S.
      • Beaudet A.
      • Paylor R.
      • De Biasi M.
      The nicotinic acetylcholine receptor subunit α5 mediates short-term effects of nicotine in vivo.
      ,
      • Wang N.
      • Orr-Urtreger A.
      • Chapman J.
      • Rabinowitz R.
      • Korczyn A.D.
      Nicotinic acetylcholine receptor α5 subunits modulate oxotremorine-induced salivation and tremor.
      ).

      Acknowledgments

      We thank Dr. Jerry A. Stitzel (Department of Integrative Physiology, University of Colorado, Boulder, CO) for providing mouse nAChR subunits. We also thank Drs. Yongchang Chang and Paul Whiteaker of the Barrow Neurological Institute for comments about the project and manuscript and for technical advice and Minoti Bhakta for assistance.

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