title: Mechanisms for picrotoxin block of alpha2 homomeric glycine receptors.
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.
authors.affiliation: Unité Mixte de Recherche, CNRS 7102, Neurobiologie des Processus Adaptatifs, Université Pierre et Marie Curie, Paris, France.
authors.forename: Dian-Shi
authors.initials: DS
authors.lastname: Wang
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Mangin
authors.forename: Gustave
authors.initials: G
authors.lastname: Moonen
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Rigo
authors.forename: Pascal
authors.initials: P
authors.lastname: Legendre
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000494:pharmacology; D007700:Kinetics; D008390:Markov Chains; D008954:Models, Biological; D010852:Picrotoxin / Q000494:pharmacology*; D018009:Receptors, Glycine / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D005998:Glycine; D010852:Picrotoxin; D018009:Receptors, Glycine; C439504:GLRA2 protein, human