Project 9. Effects of thyroid hormones on GABA(A) receptors investigated experimentally and computationally (G. Brannigan, Physics; J. V. Martin, Biology)

Background. Thyroid hormones affect the gene expression (genomic effects) in many organs and the developing brain [1] (and possibly the adult brain [2]). Hypothyroidism or hyperthyroidism in mature animals are clearly related to influences of the hormones on the central nervous system (CNS) [3, 4]. The hormones are expected to have nongenomic, neurotransmitter-like effects in adult brains [5-8]. We have documented effects of thyroid hormones on the GABAA receptor (the major receptor for the primary inhibitory neurotransmitter in the brain, ?-aminobutyric acid or GABA) [8-10]. A molecular dynamics homology model of the GABAA receptor was obtained, based on a C. elegans  glutamate-gated chloride channel (GluCl) receptor [11].  The GluCl had been crystallized around ivermectin, the anti-helminthic drug [12], and when the ivermectin was replaced in the molecular dynamics model by a L-triiodothyronine (T3, a thyroid hormone), the binding was stable in the simulation [13]. This suggests a competition for the binding of ivermectin on the GABAA receptor, supported by experiments measuring chloride currents by two-electrode voltage clamp (TEVC) in Xenopus oocytes expressing the GABAA receptor [13]. The studies to date have therefore provided evidence for a GABAA receptor binding site for T3 which may be shared with ivermectin and neurosteroids. 

Research. The research will investigate the nature of the binding site on the GABAA receptor for thyroid hormones.  The work will use a combination of computational and experimental approaches to determine the specificity of the effect.  A series of neurosteroids and thyroid hormones will be tested for docking to the site for ivermectin in the model. The compounds will include, for example, pregnenolone sulfate (PS), 5?-Pregnan-3?-ol-20-one (allopregnanolone),  3?,21-dihydroxy-5?-pregnan-20-one (THDOC),  L-triiodothyroacetic acid  (triac) and variously iodinated thyronamines. We will determine whether the compound of interest has a positive (GABA-enhancing) or negative (GABA-inhibiting) effect.  A positive modulator (e.g. ivermectin) will be paired with a negative modulator (e.g. thyroid hormone) and Schild analyses [14] will determine a competitive or non-competitive effect.  By pairing compounds of interest with appropriate counterparts, it will be possible to determine if the compounds compete for a single site on the receptor.  In this way, the site for binding of thyroid hormones will be characterized.

Student Activities. The students will use the program Autodock Vina [15] to provide automated docking of the compounds to the complete GABAA receptor model.  With VMD [16] and the CHARMM36 forcefield [17] they will measure the strength of energetic interactions between the ligand and the binding site and decompose them into contributions from different chemical groups. In the experimental laboratory, the students will surgically remove oocytes from a female Xenopus laevis frog, defolliculate the oocytes with collagenase and inject them with the cRNAs for the GABAA receptor subunits.  The oocytes will be incubated, placed in a chamber perfused with ND96 solution and impaled with two glass microelectrodes to perform all aspects of the TEVC method [10].  Subsequently, the students will perform a Schild analysis [14] of the data using the GraphPad software [18].