The glycine receptor (GlyR) is a neurotransmitter-gated chloride channel that mediates fast inhibitory neurotransmission, predominantly in the spinal cord and brain stem. Mutations of the GlyR are the major cause of hereditary hyperekplexia. Site-specific cysteine substitution followed by labeling with a fluorophore has previously been used to explore the behaviors of the hyperekplexia-related 271 (19') residue of the GlyR. However, this manipulation dramatically compromises sensitivity toward the agonist glycine and alters the pharmacological effects of various agents in manners similar to those of the hyperekplexia-causing R19'Q/L mutations, raising the question whether what is reported by the substituted and modified residue faithfully reflects what actually happens to the wild-type (WT) residue. In this study, a mechanism-rescuing second-site mutation was introduced to create a WT-mimicking GlyR (with the 19' residue cysteine substitution and modification still in place), in which the sensitivity toward glycine and pharmacological effects of various agents were restored. Further experiments revealed stark differences in the behaviors upon the various pharmacological treatments and consequently the underlying mechanisms of the 19' residue between this WT-mimicking GlyR and the GlyR without the mechanism rescue, which is correspondingly defined as the disease-type (DT)-mimicking GlyR. The data presented in this study warn generally that caution is required when attempting to deduce the behaviors of a WT residue from data based on substituted or modified residues that alter protein structure and function. Extra measures, such as rescuing mechanisms via alternative means as presented in this study, are needed to mitigate this challenge.
Keywords: Site-specific modification; glycine receptor; mechanism rescue; second-site mutation; voltage-clamp fluorometry.