O'Connor, Vincent Maurice; (1992) Characterization of strychnine binding sites in the rodent spinal cord. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The convulsant alkaloid strychnine is a selective and highly potent antagonist at postsynaptic receptor for the inhibitory neurotransmitter glycine. These properties have led to the extensive use of strychnine as a ligand to probe the postsynaptic glycine receptor. Despite the recent increased understanding of the molecular structure of this receptor protein there is still much dispute as to the nature of the interaction between glycine and strychnine. In an attempt to more clearly define this interaction the experiments described, combine the techniques of protein modification and ligand binding. These investigations also revealed a novel [3H]-strychnine binding site in the rodent CNS and attempts were made to characterize this phenomenon. The results described suggest that glycine is a fully competitive inhibitor of [3H]-strychnine binding and its reported action as a partial competitive inhibitor is an artefact of the assay conditions. The disruption of [3H]-strychnine binding by residue selective protein modifying reagents suggests some overlap in the strychnine and glycine binding sites at the receptor. Protection studies confirm this and the results are best explained by overlapping yet conformationally distinct recognition sites for strychnine and glycine. Experiments which describe protein modification and ligand protection of strychnine binding antisera highlight possible congruence in the molecular recognition at the antisera and the receptor. This is of interest in the light of proposed models of the strychnine binding site at the postsynaptic glycine receptor. Modification of spinal cord membranes by the arginine selective reagent 2,3-butanedione (BD) reveals a low affinity and high capacity [3H]-strychnine binding site which is not detectable in untreated membranes. This binding site showed a similar distribution in the CNS as the high affinity site. However, experiments using affinity purified glycine receptor and crude membrane preparations from the mutant mouse spastic indicated that the BD-induced binding site is not located on the postsynaptic glycine receptor. Competition studies revealed that [3H]-strychnine binding sites in untreated and BD-treated membranes have different structural determinants. The ability to effectively inhibit [3H]-strychnine binding to the BD-induced site by cation channel blockers is in accord with reports that strychnine can interact with various cation channels to open or block them. In addition several compounds that inhibit the BD-induced [3H]-strychnine binding can also modulate the reaction of BD with spinal cord membranes if present during the treatment, suggesting a conformational dependent modification. Upon exposure to ultraviolet light [3H]-strychnine is specifically incorporated into a low molecular weight peptide in BD-treated membranes in addition to the ligand binding subunit of the inhibitory glycine receptor, which is the only peptide photolabelled in untreated membranes. The significance of this biochemical and pharmacological characterization of this previously undescribed strychnine binding site is presently unclear. However, the uneven distribution in the CNS and the interaction with important therapeutic agents; local anaesthetics and anti-arrhythmics, indicate the possible biological importance of the novel strychnine binding site.

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