Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors*

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal γ-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of α2 homomeric GlyR is still unclear compared with that of α1 homomeric GlyR, GABAA, and GABAC 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 α2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the α2 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 α2 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 α2 GlyR.