Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABA_A Receptor

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABA_A receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed “DM compounds”) derived from the previously characterized GABA_A and the nicotinic a7 receptor modulator (E)-3furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary abc GABA_A receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-Nphenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (DG ̴ –1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the a1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate.