A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function

Dai-Fei Wu, Nidaa A. Othman, Douglas Sharp, Arjun Mahendra, Tarek Z. Deeb, Tim G. Hales (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha 7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

    Original languageEnglish
    Pages (from-to)603-615
    Number of pages13
    JournalJournal of Physiology
    Volume588
    Issue number4
    DOIs
    Publication statusPublished - 15 Feb 2010

    Keywords

    • GATED ION-CHANNEL
    • NICOTINIC ACETYLCHOLINE-RECEPTOR
    • TRYPTOPHAN-SCANNING MUTAGENESIS
    • MU-OPIOID RECEPTOR
    • CELL-SURFACE EXPRESSION
    • X-RAY-STRUCTURE
    • GENERAL-ANESTHETICS
    • GABA(A) RECEPTORS
    • M3
    • ENDOCYTOSIS

    Cite this

    Wu, Dai-Fei ; Othman, Nidaa A. ; Sharp, Douglas ; Mahendra, Arjun ; Deeb, Tarek Z. ; Hales, Tim G. / A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function. In: Journal of Physiology. 2010 ; Vol. 588, No. 4. pp. 603-615.
    @article{87bec407ee3043f0a903ebdeea0dbe10,
    title = "A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function",
    abstract = "The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha 7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.",
    keywords = "GATED ION-CHANNEL, NICOTINIC ACETYLCHOLINE-RECEPTOR, TRYPTOPHAN-SCANNING MUTAGENESIS, MU-OPIOID RECEPTOR, CELL-SURFACE EXPRESSION, X-RAY-STRUCTURE, GENERAL-ANESTHETICS, GABA(A) RECEPTORS, M3, ENDOCYTOSIS",
    author = "Dai-Fei Wu and Othman, {Nidaa A.} and Douglas Sharp and Arjun Mahendra and Deeb, {Tarek Z.} and Hales, {Tim G.}",
    year = "2010",
    month = "2",
    day = "15",
    doi = "10.1113/jphysiol.2009.181719",
    language = "English",
    volume = "588",
    pages = "603--615",
    journal = "Journal of Physiology",
    issn = "0022-3751",
    publisher = "Wiley",
    number = "4",

    }

    A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function. / Wu, Dai-Fei; Othman, Nidaa A.; Sharp, Douglas; Mahendra, Arjun; Deeb, Tarek Z.; Hales, Tim G. (Lead / Corresponding author).

    In: Journal of Physiology, Vol. 588, No. 4, 15.02.2010, p. 603-615.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function

    AU - Wu, Dai-Fei

    AU - Othman, Nidaa A.

    AU - Sharp, Douglas

    AU - Mahendra, Arjun

    AU - Deeb, Tarek Z.

    AU - Hales, Tim G.

    PY - 2010/2/15

    Y1 - 2010/2/15

    N2 - The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha 7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

    AB - The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT3) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT3B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha 7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

    KW - GATED ION-CHANNEL

    KW - NICOTINIC ACETYLCHOLINE-RECEPTOR

    KW - TRYPTOPHAN-SCANNING MUTAGENESIS

    KW - MU-OPIOID RECEPTOR

    KW - CELL-SURFACE EXPRESSION

    KW - X-RAY-STRUCTURE

    KW - GENERAL-ANESTHETICS

    KW - GABA(A) RECEPTORS

    KW - M3

    KW - ENDOCYTOSIS

    UR - http://www.scopus.com/inward/record.url?scp=77949522347&partnerID=8YFLogxK

    U2 - 10.1113/jphysiol.2009.181719

    DO - 10.1113/jphysiol.2009.181719

    M3 - Article

    VL - 588

    SP - 603

    EP - 615

    JO - Journal of Physiology

    JF - Journal of Physiology

    SN - 0022-3751

    IS - 4

    ER -