Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors

Dian-Shi Wang,Jean-Marie Mangin,Gustave Moonen,Jean-Michel Rigo,Pascal Legendre

doi:10.1074/jbc.m511022200

Abstract

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.

Highlights

Glycine and GABA3 are the main inhibitory neurotransmitters in the central nervous system
Concentration-dependent Inhibition of ␣2 Homomeric glycine receptor (GlyR) by PTX— We first analyzed the ability of PTX to inhibit GlyR activity in terms of the outside-out current evoked by glycine applications to patches containing recombinant ␣2 homomeric GlyR from Chinese hamster ovary (CHO) cells stably expressing the ␣2 GlyR subunit
We demonstrated several unexpected new features for PTX inhibition of wild-type homomeric ␣2 GlyR recorded on outside-out patches

Summary

Introduction

Glycine and GABA3 are the main inhibitory neurotransmitters in the central nervous system. The authors proposed that PTX preferentially binds to the agonist-bound conformation of the receptor and stabilized the channel in the closed state (16). This was demonstrated with GABAC receptors from isolated retinal bipolar cells and from oocytes expressing the GABAR ␳ subunit (17). We showed that PTX inhibited ␣2 homomeric GlyRs in a concentration-dependent and voltage-independent manner, and that PTX could bind to the receptor in both the open channel conformation and the fully liganded closed state. We demonstrated that PTX could be trapped at its binding site when the channel closed during glycine dissociation This complex mechanism can be predicted by a simple kinetic model in which glycine can dissociate while PTX remains bound

Methods

Results

Conclusion