Macroscopic responses of α2 GlyR to rapid glycine application to outside-out patches. (A) The current trace represents the average of 12 sweeps evoked by a 2-ms concentration jump of 10 mM glycine applied to an outside-out patch (held at −100 mV) from a HEK293 cell expressing α2 GlyRs. Recordings were performed with 10 mM Cl⁻ pipette solution. The time course of glycine application, taken from the open tip response after seal rupture, is shown above the trace. (B) Similarly, the average current obtained with a long pulse (200 ms) of 10 mM glycine (average of 20 sweeps). Note the complex desensitization phase (for this patch the decay was fitted by three components, with time constants and areas of 1.8 ms, 0.5%; 25 ms, 1.5%; and 1,300 ms, 98%). (C) Traces are average currents elicited in one outside-out patch by two to five glycine applications using 10 mM Cl⁻ pipette solution. Glycine concentrations were applied in random order and for a time sufficient to obtain a peak current. (D) Concentration–response curve for glycine obtained from pooled data from outside-out currents as those in C (refer to Materials and methods for details). Currents were normalized to the fitted maximum for each experiment, and the pooled data were fitted with the Hill equation (n = 6).