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Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) ?3 subunits in enhanced functional expression of mouse ?6?2?3- and ?6?4?3-nAChRs.

ABSTRACT: Functional heterologous expression of naturally expressed mouse ?6*-nicotinic acetylcholine receptors (m?6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid m?6m?4h?3- (? 5-8-fold) or WT m?6m?4m?3-nAChRs (? 2-fold) yielded higher function than m?6m?4-nAChRs. Function was not detected when m?6 and m?2 subunits were expressed together or in the additional presence of h?3 or m?3 subunits. However, function emerged upon expression of m?6m?2m?3(V9'S)-nAChRs containing ?3 subunits having gain-of-function V9'S (valine to serine at the 9'-position) mutations in transmembrane domain II and was further elevated 9-fold when h?3(V9'S) subunits were substituted for m?3(V9'S) subunits. Studies involving WT or gain-of-function chimeric mouse/human ?3 subunits narrowed the search for domains that influence functional expression of m?6*-nAChRs. Using h?3 subunits as templates for site-directed mutagenesis studies, substitution with m?3 subunit residues in extracellular N-terminal domain loops "C" (Glu(221) and Phe(223)), "E" (Ser(144) and Ser(148)), and "?2-?3" (Gln(94) and Glu(101)) increased function of m?6m?2*- (? 2-3-fold) or m?6m?4* (? 2-4-fold)-nAChRs. EC50 values for nicotine acting at m?6m?4*-nAChR were unaffected by ?3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops ?2-?3 and E) interfaces of ?3 subunits are some of the molecular impediments for functional expression of m?6m?2?3- or m?6m?4?3-nAChRs.

SUBMITTER: Dash B

PROVIDER: S-EPMC4192487 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) β3 subunits in enhanced functional expression of mouse α6β2β3- and α6β4β3-nAChRs.

Dash Bhagirathi B Li Ming D MD Lukas Ronald J RJ

The Journal of biological chemistry 20140715 41

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where "*" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼ 5-8-fold) or WT mα6mβ4mβ3-nAChRs (∼ 2-fold) yielded higher funct ...[more]

PMID: 25028511

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Project description: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.

Dataset Information

Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) ?3 subunits in enhanced functional expression of mouse ?6?2?3- and ?6?4?3-nAChRs.

Publications

Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) β3 subunits in enhanced functional expression of mouse α6β2β3- and α6β4β3-nAChRs.

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