Molecular characterization of agonists that bind to an insect GABA receptor

Abstract

Ionotropic GABA receptors are widely distributed throughout the vertebrate and invertebrate central nervous system (CNS) where they mediate inhibitory neurotransmission. One of the most widely studied insect GABA receptors is constructed from RDL (resistance to dieldrin) subunits from Drosophila melanogaster. The aim of this study was to determine critical features of agonists binding to RDL receptors using in silico and experimental data. Partial atomic charges and dipole separation distances of a range of GABA analogues were calculated, and the potency of the analogues was determined using RDL receptors expressed in Xenopus oocytes. These data revealed functional agonists require an ammonium group and an acidic group with an optimum separation distance of approximately 5 A. To determine how the agonists bind to the receptor, a homology model of the extracellular domain was generated and agonists were docked into the binding site. The docking studies support the requirements for functional agonists and also revealed a range of potential interactions with binding site residues, including hydrogen bonds and cation-pi interactions. We conclude that the model and docking procedures yield a good model of the insect GABA receptor binding site and the location of agonists within it.

Figure 1

(A) RDL extracellular domain dimer. Extracellular loops A−F that form the agonist binding site region in related Cys-loop receptors are labeled. (B) Sequence alignment of extracellular domains of RDL and homologous Cys-loop receptor subunits (human GABA_A α1, human GABA_A β2, mouse 5-HT₃A, and AChBP).

Figure 2

(A) Electrophysiological traces showing maximal currents elicited by agonists tested at RDL receptors: GABA (100 μM), TACA (300 μM), isoguvacine (600 μM), β-alanine (10 mM), and taurine (10 mM). (B) Concentration response curves showing responses elicited by GABA and analogues at wild-type RDL receptors expressed in Xenopus oocytes: muscimol > GABA > TACA > isoguvacine > THIP > 5-AV > β-alanine > taurine. Data are means ± SEM; n ≥ 3.

Figure 3

Chemical structures of GABA analogues examined in this study.

Figure 4

Docking of GABA and active analogues (β-alanine, TACA, 5-AV, taurine, isoguvacine, muscimol, and THIP) into the RDL binding site. Analogues docked with the ammonium group located deep in the cleft formed between loop B and loop C aromatic residues. The carboxylate moiety or equivalent substituent was predicted to be located facing toward Arg111.