The seizure-inducing plastic explosive RDX inhibits the α1β2γ2 GABAA receptor

Abstract

Objective: Royal demolition explosive (RDX) can induce seizures in wildlife and humans following release into the environment or after voluntary consumption. During the Vietnam War, RDX intoxication was the most common cause of generalized seizures in US service personnel, and in some sections of the armed forces, eating of RDX has continued as "a dare" to this day. After its mechanism of action was long unknown, RDX was recently shown to be a GABA_A receptor antagonist. We here determined the GABA_A receptor subtype-selectivity of RDX and mapped its functional binding site.

Methods: We used whole-cell patch-clamp to determine the potency of RDX on 10 recombinantly expressed GABA_A receptors and mapped the RDX binding site using a combination of Rosetta molecular modeling and site-directed mutagenesis.

Results: RDX was found to reversibly inhibit the α1β2γ2 GABA_A receptor with an IC₅₀ of 23 μmol/L (95% CI 15.1-33.3 μmol/L), whereas α4 and α6 containing GABA_A receptor combinations were 4-10-fold less sensitive. RDX is binding to the noncompetitive antagonist (NCA) site in the pore. In a molecular model based on the cryo-EM structure of the resting state of the α1β2γ2 receptor, RDX forms two hydrogen bonds with the threonines at the T6' ring and makes hydrophobic interactions with the valine and alanine in 2' position of the α1 or β2 subunits.

Interpretation: Our findings characterize the mechanism of action of RDX at the atomistic level and suggest that RDX-induced seizures should be susceptible to treatment with GABA_A modulating drugs such as benzodiazepines, barbiturates, propofol, or neurosteroids.

Figure 1

Concentration–response curves and IC₅₀ values for RDX‐mediated inhibition of currents evoked by EC₉₀ GABA for α1 and α2 (left) or α4 and α6 (right) containing GABA_A receptors. Data points are mean ± SD from 3 to 8 independent recordings. IC₅₀ values are presented with 95% confidence intervals. The EC₉₀ GABA concentration was 110 μmol/L for α1β1γ2L, 100 μmol/L for α1β2γ2L, 60 μmol/L for α1β3γ2L, 40 μmol/L for α1β2, 50 μmol/L for α2β3γ2L, 10 μmol/L for α4β3γ2L, 10 μmol/L for α4β3γ1, 5 μmol/L for α4β3δ, 15 μmol/L for α6β1γ2L, and 10 μmol/L for α6β3γ2L. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

RDX is a reversible, noncompetitive inhibitor of the α1β2γ2L GABA_A receptor. (A) Example recording showing that RDX is reversible on washout. Currents were elicited by 100 μmol/L GABA. (B) Comparison of the effects of bicuculline (5 μmol/L) and RDX (100 μmol/L) on the GABA concentration–response curve. Data points are mean ± SD from 3 to 8 independent recordings. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

RDX enhances GABA effects at low concentrations. (A) Example recording showing that 1 μmol/L of RDX enhances chloride currents elicited by 100 μmol/L GABA while 100 μmol/L RDX inhibits currents. (B) Scatterplots showing the percentage of current activation by 0.5, 1, and 2 μmol/L of RDX (n = 8 to 13 cells, error bars are ±SD). (C) Comparison of the effects of allopregnanolone (75 nmol/L) and RDX (1 μmol/L) on the GABA concentration–response curve of the α1β2γ2L GABA_A receptor. Data points are mean ± SD from 3 to 8 independent recordings. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

The RDX binding site. (A) Molecular model of the RDX binding site in the pore of the α1β2γ2 receptor identified of RosettaLigand. The receptor is color‐coded as follows: α1 (blue), β2 (red), and γ2 (yellow). RDX is shown as a green space‐filled model. One α1 subunit is removed to allow a view of the permeation pathway. (B and C) Closeup views of the two alternative, low‐energy binding poses of RDX at T6’ ring. RDX is shown in stick representation, hydrogen bonds are shown in green. (D) Site‐directed mutagenesis of the α1β2γ2L receptor. Percentage of current blocked by 100 μmol/L RDX (mean ± SD from n = 5–8 cells per mutant) was analyzed with one‐way ANOVA followed by Dunnett's test to compare the means to the WT control and to correct for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Representative examples from the mutagenesis. (A) The β2A2’M mutant is insensitive to RDX. Currents were elicited by 100 μmol/L GABA. (B) RDX concentration–response curves for the wild‐type α1β2γ2L receptor and the α1T6’M, β2A2’M, and γ2S2’G mutants. Data points are mean ± SD from 3 to 8 independent recordings. [Colour figure can be viewed at wileyonlinelibrary.com]