ABSTRACT: 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 h?6*-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 h?6 subunit to probe for their effect on the functional expression of h?6*-nAChRs.N-terminal ?-helix (Asp57); complementary face/inner ?-fold (Arg87 or Asp92) and principal face/outer ?-fold (Ser156 or Asn171) residues in the h?6 subunit are crucial for functional expression of the h?6*-nAChRs as variations in these residues reduce or abrogate the function of h?6h?2*-, h?6h?4- and h?6h?4h?3-nAChRs. While variations at residues Ser43 or Asn46 (both in N-terminal ?-helix) in h?6 subunit reduce h?6h?2*-nAChRs function those at residues Arg96 (?2-?3 loop), Asp199 (loop F) or Ser233 (?10-strand) increase h?6h?2*-nAChR function. Similarly substitution of NTD ?-helix (Asn46), loop F (Asp199), loop A (Ala112), loop B (Ala184), or loop C (Ile226) residues in h?6 subunit increase the function of h?6h?4-nAChRs. All other variations in h?6 subunit do not affect the function of h?6h?2*- and h?6h?4*-nAChRs. Incorporation of nAChR h?3 subunits always increase the function of wild-type or variant h?6h?4-nAChRs except for those of h?6(D57N, S156R, R87C or N171K)h?4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in h?6 subunit drive the h?6h?4h?3-nAChRs into a nonfunctional state as at spontaneously open h?6(D57N, S156R or N171K)h?4h?3V9'S-nAChRs (V9'S; transmembrane II 9' valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of h?6h?4- and/or h?6h?4h?3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in h?6 subunit.Hence investigating functional consequences of natural variations in nAChR h?6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of h?6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in h?6 subunit that compromise h?6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.
