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

Abstract

Background: Functional heterologous expression of naturally-expressed and apparently functional mammalian 6*-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 6 subunit in the functional expression of h6*-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 h6 subunit to probe for their effect on the functional expression of h6*-nAChRs. Results: N-terminal -helix (Asp57); complementary face/inner β-fold (Arg87 or Asp92) and principal face/outer β-fold (Ser156 or Asn171) residues in the h6 subunit are crucial for functional expression of the h6*-nAChRs as variations in these residues reduce or abrogate the function of h6hβ2*-, h6hβ4- and h6hβ4hβ3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal -helix) in h6 subunit reduce h6hβ2*-nAChRs function those at residues Arg96 (β2-β3 loop), Asp199 (loop F) or Ser233 (β10-strand) increase h6hβ2*-nAChR function. Similarly substitution of NTD -helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in h6 subunit increase the function of h6hβ4-nAChRs. All other variations in h6 subunit do not affect the function of h6hβ2*- and h6hβ4*-nAChRs. Incorporation of nAChR hβ3 subunits always increase the function of wild-type or variant h6hβ4-nAChRs except for those of h6(D57N, S156R, R87C or N171K)hβ4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in h6 subunit drive the h6hβ4hβ3- nAChRs into a nonfunctional state as at spontaneously open h6(D57N, S156R or N171K)hβ4hβ3V9'S-nAChRs (V9'S; transmembrane II 9' valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of h6hβ4- and/or h6hβ4hβ3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in h6 subunit. Conclusions: Hence investigating functional consequences of natural variations in nAChR h6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of h6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in h6 subunit that compromise h6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine. © 2014 Dash and Li; licensee BioMed Central Ltd.