Loop G in the GABAA receptor α1 subunit influences gating efficacy

Daniel T. Baptista-Hon, Simona Gulbinaite, Tim G. Hales (Lead / Corresponding author)

    Research output: Contribution to journalArticle

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    Abstract

    The modification of Cys residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G impairs activation of α1β2γ2 receptors by GABA and propofol (Baptista-Hon et al. 4). While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub-maximal, but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline. This article is protected by copyright. All rights reserved.

    Original languageEnglish
    Pages (from-to)1725-1741
    Number of pages16
    JournalJournal of Physiology
    Volume595
    Issue number5
    Early online date16 Dec 2016
    DOIs
    Publication statusPublished - 1 Mar 2017

    Fingerprint

    Propofol
    GABA-A Receptors
    gamma-Aminobutyric Acid
    GABA Receptors
    Bicuculline
    Immobilization
    Mutation

    Keywords

    • Cys-loop receptors
    • mutagenesis
    • spontaneous gating
    • pentameric ligand-gated ion channels

    Cite this

    @article{932b483c6fa6440e939b5160e5621411,
    title = "Loop G in the GABAA receptor α1 subunit influences gating efficacy",
    abstract = "The modification of Cys residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G impairs activation of α1β2γ2 receptors by GABA and propofol (Baptista-Hon et al. 4). While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub-maximal, but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline. This article is protected by copyright. All rights reserved.",
    keywords = "Cys-loop receptors, mutagenesis, spontaneous gating, pentameric ligand-gated ion channels",
    author = "Baptista-Hon, {Daniel T.} and Simona Gulbinaite and Hales, {Tim G.}",
    note = "Funding – This work was supported by funding from Tenovus Scotland.",
    year = "2017",
    month = "3",
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    doi = "10.1113/JP273752",
    language = "English",
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    Loop G in the GABAA receptor α1 subunit influences gating efficacy. / Baptista-Hon, Daniel T.; Gulbinaite, Simona; Hales, Tim G. (Lead / Corresponding author).

    In: Journal of Physiology, Vol. 595, No. 5, 01.03.2017, p. 1725-1741.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Loop G in the GABAA receptor α1 subunit influences gating efficacy

    AU - Baptista-Hon, Daniel T.

    AU - Gulbinaite, Simona

    AU - Hales, Tim G.

    N1 - Funding – This work was supported by funding from Tenovus Scotland.

    PY - 2017/3/1

    Y1 - 2017/3/1

    N2 - The modification of Cys residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G impairs activation of α1β2γ2 receptors by GABA and propofol (Baptista-Hon et al. 4). While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub-maximal, but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline. This article is protected by copyright. All rights reserved.

    AB - The modification of Cys residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABAA α1 subunit loop G impairs activation of α1β2γ2 receptors by GABA and propofol (Baptista-Hon et al. 4). While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C and T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub-maximal, but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABAA receptor α1 subunit's β1 strand during agonist dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline. This article is protected by copyright. All rights reserved.

    KW - Cys-loop receptors

    KW - mutagenesis

    KW - spontaneous gating

    KW - pentameric ligand-gated ion channels

    U2 - 10.1113/JP273752

    DO - 10.1113/JP273752

    M3 - Article

    VL - 595

    SP - 1725

    EP - 1741

    JO - Journal of Physiology

    JF - Journal of Physiology

    SN - 0022-3751

    IS - 5

    ER -