Molecular Pharmacology, Vol 10, 790-809, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Strychnine Binding in Rat Spinal Cord Membranes Associated with the Synaptic Glycine Receptor: Cooperativity of Glycine Interactions

¹ Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The saturable binding of [³H]strychnine to synaptic membrane fractions of the rat spinal cord appears to involve an interaction with the synaptic receptor sites for the neurotransmitter actions of glycine. Binding exhibits a dissociation constant for strychnine of 2.6-4 nM and an EC₅₀ for glycine inhibition of strychnine binding of 25 µM. Association kinetics is bimolecular, with a rate constant of 1.0 x 10⁷ M^-1 sec^-1, while dissociation of the strychnine-receptor complex is first-order, with a rate constant of 1.54 x 10^-2 sec^-1. The dissociation constant (k₂/k₁) of 1.54 nM is similar to that obtained from equilibrium data. Monovalent cations increase specific and decrease nonspecific strychnine binding, while divalent cations are without effect. Detergents such as deoxycholate and Triton X-100 decrease receptor binding in concentrations which solubilize membrane protein. Displacement curves of [³H]strychnine by glycine indicate cooperative interactions with a Hill coefficient of 1.7, whereas the Hill coefficient for displacement of [³H]strychnine by unlabeled strychnine is 1.0. Diazonium tetrazole and acetic anhydride preferentially inhibit displacement of [³H]strychnine binding by glycine and reduce the Hill coefficient of glycine displacement curves. Guanidine, N-ethylmaleimide, and increases in pH also lower the Hill coefficient for glycine displacement. Diazonium tetrazole slows the dissociation of [³H]strychnine elicited by excess glycine without altering dissociation produced by excess unlabeled strychnine. Glycine and strychnine appear to bind to distinct sites which interact in a cooperative fashion.

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