INTERACTIONS OF GLYCINE AND STRYCHNINE WITH THEIR RECEPTOR RECOGNITION SITES IN MOUSE SPINAL-CORD

Interactions between the inhibitory neurotransmitter glycine and its r eceptor antagonist strychnine have been studied in mouse spinal cord m embranes and both agents employed to protect against residue selective modifying reagents in order to identify contact residues for ligand b inding. Glycine was found to behave as a full competitive inhibitor of [H-3]-strychnine binding, provided that precautions were taken to pre vent radioligand binding to the glass-fibre filters used to terminate the assays. Hill coefficients for the glycine inhibition of [H-3]-stry chnine binding were not significantly different from one, indicating a lack of cooperative interactions. For the protection experiments, N-b romosuccinimide, tetranitromethane, diethylpyrocarbonate and 2,3-butan edione were used under conditions selective for tryptophan, tyrosine, histidine and arginine residues, respectively. Of these reagents, N-br omosuccinimide, tetranitromethane and diethylpyrocarbonate caused a de crease in total [H-3]-strychnine binding without affecting the ability of unlabelled strychnine to compete. In contrast, the same reagents d isrupted the ability of glycine to inhibit [H-3]-strychnine binding. t he presence of either excess glycine (10(-2) M) or strychnine (10(-4) M) during the above treatments was found to prevent the decrease in to tal and strychnine-specific [H-3]-strychnine binding. However, only in the case of diethylpyrocarbonate treatment were both agonist and anta gonist able to protect against the loss of glycine-specific [H-3]-stry chnine binding. The reagent 2,3-butanedione caused an increase in tota l and strychnine-specific [H-3]-strychnine binding (which we have show n elsewhere to be at a site unrelated to the inhibitory glycine recept or). When the above protein modifying reagents were applied under the same conditions to specific strychnine binding antibodies, all four ca used significant decreases in subsequent [H-3]-strychnine binding. Str ychnine was found to afford significant protection of the antibodies a gainst N-bromosuccinimide, tetranitromethane and 2,3-butanedione, but not against diethylpyrocarbonate. Our results suggest that glycine and strychnine compete at overlapping but conformationally distinct sites on the receptor. Tyrosine, tryptophan. histidine and arginine residue s are implicated as strychnine contact residues with a shared role for histidine in the recognition of glycine. Copyright (C) 1996 Elsevier Science Ltd.