Abstract
Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.
Highlights
The ionotropic γ-aminobutyric acid (GABA) type A receptor (GABAA) is a primary transducer of inhibitory signaling in the central nervous system
Over the course of a 200 ns Molecular dynamics (MD) simulation of T3 bound to a GABAA receptor model in a hydrated lipid bilayer, we find that T3 is stable in four intersubunit sites over these timescales, with spontaneous rebinding observed for the fifth interface
The inhibiting effect of T3 (0.1 μM- 100 μM) on GABAA receptor stimulation by 10 μM GABA is shown in Fig 2A, with a representative trace indicating a significant reduction in the response of GABA in the presence of 10 μM T3 (Fig 2A, inset)
Summary
The ionotropic γ-aminobutyric acid (GABA) type A receptor (GABAA) is a primary transducer of inhibitory signaling in the central nervous system. We have previously proposed [11] that the thyroid hormone, triiodothyronine (T3) modulates the GABAA receptor via a mechanism similar to that of neurosteroids. Such modulation has potential physiological significance as a non-genomic mechanism through which T3 exerts its significant effects on sleep and mood in adulthood [12,13,14,15]. T3 inhibits the activity of GABA-gated chloride currents on recombinant GABAA receptors expressed in human embryonic kidney-293 cells and Xenopus oocytes at lower concentrations [16], but activates at concentrations beyond 30 μM [16,17]
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