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

作者：Dash Bhagirathi; Li Ming D^*

来源：Molecular Brain, 2014, 7(1): 35.

DOI：10.1186/1756-6606-7-35

• Electrophysiology
• Ion channels
• Nicotinic acetylcholine receptor
• Receptor structure-function
• Single nucleotide variation

Background: Functional heterologous expression of naturally-expressed and apparently functional mammalian alpha 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 alpha 6 subunit in the functional expression of h alpha 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 alpha 6 subunit to probe for their effect on the functional expression of h alpha 6*-nAChRs.
Results: N-terminal alpha-helix (Asp(57)); complementary face/inner beta-fold (Arg(87) or Asp(92)) and principal face/outer beta-fold (Ser(156) or Asn(171)) residues in the h alpha 6 subunit are crucial for functional expression of the h alpha 6*-nAChRs as variations in these residues reduce or abrogate the function of h alpha 6h beta 2*-, h alpha 6h beta 4-and h alpha 6h beta 4h beta 3-nAChRs. While variations at residues Ser(43) or Asn(46) (both in N-terminal alpha-helix) in h alpha 6 subunit reduce h alpha 6h beta 2*-nAChRs function those at residues Arg(96) (beta 2-beta 3 loop), Asp(199) (loop F) or Ser(233) (beta 10-strand) increase h alpha 6h beta 2*-nAChR function. Similarly substitution of NTD alpha-helix (Asn(46)), loop F (Asp(199)), loop A (Ala(112)), loop B (Ala(184)), or loop C (Ile(226)) residues in h alpha 6 subunit increase the function of h alpha 6h beta 4-nAChRs. All other variations in h alpha 6 subunit do not affect the function of h alpha 6h beta 2*-and h alpha 6h beta 4*-nAChRs. Incorporation of nAChR h beta 3 subunits always increase the function of wild-type or variant h alpha 6h beta 4-nAChRs except for those of h alpha 6(D57N, S156R, R87C or N171K) h beta 4-nAChRs. It appears Asp57Lys, Ser156Arg or Asn171Lys variations in h alpha 6 subunit drive the h alpha 6h beta 4h beta 3-nAChRs into a nonfunctional state as at spontaneously open h alpha 6(D57N, S156R or N171K) h beta 4h beta 3(V9'S)-nAChRs (V9'S; transmembrane II 9' valine-to-serine mutation) agonists act as antagonists. Agonist sensitivity of h alpha 6h beta 4-and/or h alpha 6h beta 4h beta 3-nAChRs is nominally increased due to Arg96His, Ala184Asp, Asp199Tyr or Ser233Cys variation in h alpha 6 subunit.
Conclusions: Hence investigating functional consequences of natural variations in nAChR h alpha 6 subunit we have discovered additional bases for cell surface functional expression of various subtypes of h alpha 6*-nAChRs. Variations (Asp57Asn, Arg87Cys, Asp92Glu, Ser156Arg or Asn171Lys) in h alpha 6 subunit that compromise h alpha 6*-nAChR function are expected to contribute to individual differences in responses to smoked nicotine.

• 出版日期2014-5-2