Analysis of rare variations reveals roles of amino acid residues in the N-terminal extracellular domain of nicotinic acetylcholine receptor (nAChR) alpha6 subunit in the functional expression of human alpha6*-nAChRs.

Bhagirathi Dash,Ming D Li

doi:10.1186/1756-6606-7-35

Abstract

BackgroundFunctional heterologous expression of naturally-expressed and apparently functional mammalian α6*-nicotinic acetylcholine receptors (nAChRs; where ‘*’ indicates presence of additional subunits) has been difficult. Here we wanted to investigate the role of N-terminal domain (NTD) residues of human (h) nAChR α6 subunit in the functional expression of hα6*-nAChRs. To this end, instead of adopting random mutagenesis as a tool, we used 15 NTD rare variations (i.e., Ser43Pro, Asn46Lys, Asp57Asn, Arg87Cys, Asp92Glu, Arg96His, Glu101Lys, Ala112Val, Ser156Arg, Asn171Lys, Ala184Asp, Asp199Tyr, Asn203Thr, Ile226Thr and Ser233Cys) in nAChR hα6 subunit to probe for their effect on the functional expression of hα6*-nAChRs.ResultsN-terminal α-helix (Asp57); complementary face/inner β-fold (Arg87 or Asp92) and principal face/outer β-fold (Ser156 or Asn171) residues in the hα6 subunit are crucial for functional expression of the hα6*-nAChRs as variations in these residues reduce or abrogate the function of hα6hβ2*-, hα6hβ4- and hα6hβ4hβ3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal α-helix) in hα6 subunit reduce hα6hβ2*-nAChRs function those at residues Arg96 (β2-β3 loop), Asp199 (loop F) or Ser233 (β10-strand) increase hα6hβ2*-nAChR function. Similarly substitution of NTD α-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in hα6 subunit increase the function of hα6hβ4-nAChRs. All other variations in hα6 subunit do not affect the function of hα6hβ2*- and hα6hβ4*-nAChRs. Incorporation of nAChR hβ3 subunits always increase the function of wild-type or variant hα6hβ4-nAChRs except for those of hα6(D57N, S156R, R87C or N171K)hβ4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in hα6 subunit drive the hα6hβ4hβ3-nAChRs into a nonfunctional state as at spontaneously open hα6(D57N, S156R or N171K)hβ4hβ3V9’S-nAChRs (V9’S; transmembrane II 9’ valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of hα6hβ4- and/or hα6hβ4hβ3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in hα6 subunit.ConclusionsHence investigating functional consequences of natural variations in nAChR hα6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of hα6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in hα6 subunit that compromise hα6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.

Highlights

Functional heterologous expression of naturally-expressed and apparently functional mammalian α6*-nicotinic acetylcholine receptors has been difficult
Bioinformatic analyses indicate possible functional consequences of rare variations in nicotinic acetylcholine receptor (nAChR) α6 subunits Information retrieved from NCBI dbSNP database, NHLBI Grand Opportunity Exome Sequencing Project (ESP), multiple protein sequence alignment of human nAChR α subunits, multiple protein sequence alignment of nAChR α6 subunits from various model organisms and homology model of nAChR hα6 subunit (Figures 1 and 2) are combined to present an overview of the characteristics of the 15 single nucleotide variations (SNVs) evaluated for their effect on function of hα6*-nAChRs (Table 1)
NAChR hα6 subunit amino acid (AA) undergoing variation are fully conserved (Asn46, Asp57, Arg87, Asp92, Glu101, Ser156, Asn171, Ala184, Ile226 and Ser233), strongly conserved (Ala112 and Asp199), weakly conserved (Asn203) and non-conserved in an alignment of nAChR α6 subunits from various organisms [Figure 1B]. These results indicate that nAChR hα6 subunit AAs at positions 87 (Arg), 92 (Asp), 156 (Ser), and 171 (Asn) are conserved in both human nAChR α subunits and nAChR α6 subunits from other organisms

Summary

Introduction

Functional heterologous expression of naturally-expressed and apparently functional mammalian α6*-nicotinic acetylcholine receptors (nAChRs; where ‘*’ indicates presence of additional subunits) has been difficult. The β-strands connect to each other via loops: they are known as loops A, B and C in the principal or positive (+) face and loops D, E and F in the complementary or negative (−) face (Figures 1 and 2). These loops and additional residues in the NTD of participating α and β subunits form the ligand-binding site for nAChRs. The β6-β7 loop has a pair of disulfide-bonded cysteines separated by 13 residues that form the cysteine-loop (i.e., cys-loop) motif and it is essential for nAChR assembly and channel gating [6,7]

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