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Abstract

The activation and desensitization kinetics of the human α₁-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-μs time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and β-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k₊₁ ∼10⁷ M⁻¹ s⁻¹, and dissociation, k₋₁ = 1900 s⁻¹; 2) the rates of receptor-channel opening, k_op = 2200 s⁻¹, and closing, k_cl = 38 s−1; 3) the receptor desensitization rate, α = 0.45 s⁻¹; 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n ≥ 3; and 5) the maximum receptor-channel open probability, p₀ > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.