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(+)-Catharanthine potentiates the GABA_A receptor by binding to a transmembrane site at the β(+)/α(-) interface near the TM2-TM3 loop.

ABSTRACT: (+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABA_AR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABA_AR 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 GABA_ARs 2.4-3.5 times more efficaciously than at β1-containing GABA_ARs. The activity of (+)-catharanthine is reduced by the β2(N265S) mutation that decreases GABA_AR 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 GABA_AR 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.

SUBMITTER: Arias HR

PROVIDER: S-EPMC9178925 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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(+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the β(+)/α(-) interface near the TM2-TM3 loop.

Arias Hugo R HR Borghese Cecilia M CM Germann Allison L AL Pierce Spencer R SR Bonardi Alessandro A Nocentini Alessio A Gratteri Paola P Thodati Thanvi M TM Lim Natalie J NJ Harris R Adron RA Akk Gustav G

Biochemical pharmacology 20220315

(+)-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. Electrophys ...[more]

PMID: 35304861

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Project description:The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA(A) and GABA(B) receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA(A) receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA(A) receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA(A) receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA(A) agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA(A) antagonist bicuculline, implying a role for GABA(A) receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA(A) receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.

Dataset Information

(+)-Catharanthine potentiates the GABA_A receptor by binding to a transmembrane site at the β(+)/α(-) interface near the TM2-TM3 loop.

Publications

(+)-Catharanthine potentiates the GABA<sub>A</sub> receptor by binding to a transmembrane site at the β(+)/α(-) interface near the TM2-TM3 loop.

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