Abstract
(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at β2/3-containing GABAARs 2.4-3.5 times more efficaciously than at β1-containing GABAARs. The activity of (+)-catharanthine is reduced by the β2(N265S) mutation that decreases GABAAR potentiation by loreclezole, but not by the β3(M286C) or α1(Q241L) mutations that reduce receptor potentiation by R(+)-etomidate or neurosteroids, respectively. Competitive functional experiments indicated that the binding site for (+)-catharanthine overlaps that for loreclezole, but not those for R(+)-etomidate or potentiating neurosteroids. Molecular docking experiments suggested that (+)-catharanthine binds at the β(+)/α(-) intersubunit interface near the TM2-TM3 loop, where it forms H-bonds with β2-D282 (TM3), β2-K279 (TM2-TM3 loop), and β2-N265 and β2-R269 (TM2). Site-directed mutagenesis experiments supported the in silico results, demonstrating that the K279A and D282A substitutions, that lead to a loss of H-bonding ability of the mutated residue, and the N265S mutation, impair the gating efficacy of (+)-catharanthine. We infer that (+)-catharanthine potentiates the GABAAR through several H-bond interactions with a binding site located in the β(+)/α(-) interface in the transmembrane domain, near the TM2-TM3 loop, where it overlaps with loreclezole binding site.