Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA-A Receptors

Abstract

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α₂β₂γ₂ and α₂β₂γ₂). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.

Keywords: heterologous expression; human; pharmacology; phytopharmaceuticals.

Figure 1

(A) Alignment of Ap-RDL1 (AXY92182.1), Am-RDL (AJE68941.1), Vd-RDL1 (AVY53069.1), RDL2 (AYR04938.1) and RDL3 (AVY53071.1) amino acid sequences. Loops involved in GABA binding (LpA-F) are boxed in red, transmembrane segments (TM1-4) are boxed in blue. Amino acids that have been shown to be involved in GABA binding are underlined with a brown square and those in non-competitive antagonists binding with a green square. The location of the C->R substitution in our Ap-RDL1 clone is boxed in purple. (B) Enlargement of the TM1-TM2 sequence alignment with key amino acids for non-competitive antagonists at 2′, 6′ and 9′ locations bolded in red. Human sequences for α₂, α₄, β₂ and γ₂ GABA-A receptor subunits are also presented.

Figure 2

Biophysical characterization of the (Ap-RDL1 receptor expressed in X laevis oocytes. (A) Current trace showing the response of Ap-RDL1 to increasing concentrations of GABA from 0.1 µM to 1 mM at a membrane potential of −60 mV. (B) Full dose–response curve of Ap-RDL1 for GABA. The smooth line represents the best fit to a logistic function. See Table 1 for EC₅₀ and p values. (C) Representative current traces showing the response of the Ap-RDL1 to voltage ramps from −60 to +60 mV in NaCl, TEACl or Na acetate solutions. The calculated relative permeability Pacetate/Pchloride was 0.12 ± 0.02, n = 6. (D-top) Single-channel traces recorded in cell-attached patches of oocytes expressing the RDL1 GABAr subunit. The pipette solution contained ND and GABA at 100 µM, and the external solution was OSO-100 (see methods). Note the presence of (at least) 3 channels under the patch. (D-bottom) The amplitude histogram of the trace is shown with the value of the amplitude of each peak deduced from the multi-Gaussian fit (red curve). (E) Single-channel current-voltage curve obtained at different voltages from 6 patches as shown in (D-top) The single-channel conductance is deduced from a linear regression through the experimental points: 26 ± 4 pS.

Figure 3

Dose–response curves for GABA of seven GABA-A receptors: RDL of A. pisum and A. mellifera (A) RDL1, RDL2 and RDL3 of V. destructor (B), and α₂β₂γ₂ and α₄β₂γ₂ of H. sapiens (C). Left exemplar current traces from 1 type of receptor, right full dose–response curves of the seven GABAr types. The EC₅₀ are summarized in Supplementary Table S1.

Figure 4

Dose–response curves for fipronil obtained for the seven GABA-A receptors: RDL from A. Pisum and A. mellifera (A) RDL1, RDL2 and RDL3 from V. destructor (B), and α₂β₂γ₂ and α₄β₂γ₂ GABA-A receptors from H. sapiens (C). (Left), exemplar current traces from 1 type of receptor, (Right), full dose–response curves. The IC₅₀ are summarized in Supplementary Table S1.

Figure 5

Dose–response curves for picrotoxin obtained for the seven GABA-A receptors RDL from A. pisum and A. mellifera (A), RDL1, RDL2 and RDL3 from V. destructor (B), and α₂β₂γ₂ and α₄β₂γ₂ from H. Sapiens (C). (Left): representative current traces from 1 type of receptor; (Right): full dose–response curves. The IC₅₀ are summarized in Supplementary Table S1.

Figure 6

(A) Dose–response curves for dieldrin in conditions similar to Figure 4 and Figure 5 (single and simultaneous application of GABA and each dieldrin concentration) obtained for the seven GABA-A receptors: RDL fom A. pisum and A. mellifera, RDL1, RDL2 and RDL3 from V. destructor, and α₂β₂γ₂ and α₄β₂γ₂ from H. sapiens. (B) Representative current traces of Ap-RDL1 in response to repetitive application of GABA (50 µM) before and during continuous perfusion of dieldrin (1 µM). (C) The time course of the inhibition by dieldrin of these GABA-induced current amplitudes ((Rel.Cur.) can be approximated by a single exponential that allows to extract Rel.B (relative current block, see Materials and Methods) and a time constant Tau-Block. (D) The fit of the kinetics of the response of seven GABA-A receptors: RDL of A. Pisum and A. mellifera, RDL1, RDL2 and RDL3 of V. destructor, and α₂β₂γ₂ and α₄β₂γ₂ of H. sapiens to dieldrin gave Tau-Block and Rel.B for each receptor (see Figure 7).

Figure 7

Tau-Block (Left) and Rel.B (Right) values for the inhibition by dieldrin of each RDL receptor from A. pisum and A. mellifera, RDL1, RDL2 and RDL3 from V. destructor, and α₂β₂γ₂ and α₄β₂γ₂ from H. sapiens deduced from the mono-exponential fit of the responses shown in Figure 6D.

Figure 8

Radar graph summarizing the pharmacological data obtained with the different GABA-A receptors during this study. GABA (EC₅₀ in µM and yellow), fipronil (IC₅₀ in µM and blue), picrotoxin (IC₅₀ in µM and orange) and dieldrin (Tau-block in seconds and in green) are shown; for values see Supplementary Table S1.