Figure 2: ECl in WT and mutant KCC2-expressing HEK293-GlyRα1 cells.
(A) Representative traces of GlyR currents in cells co-transfected with two different vectors encoding only EGFP and DsRed (Mock), WT-KCC2 (WT), two KCC2 mutants expressed in individuals 1 and 2 (E50_Q93del & A191V), and mutants in individual 3 (S323P & M415V). Currents were recorded under the gramicidin-perforated voltage-clamp condition. Upper traces indicate membrane voltage (Vm) changes. The holding voltage was −40 mV. Two 1-s voltage ramps from −80 to −10 mV were applied before and during bath application of 100 μM glycine. Middle traces show membrane current (Im) responses. The humps of GlyR currents were generated during glycine application at the holding voltage of −40 mV, and the current responses to voltage ramps were generated before and during the humps. Note that the current levels immediately before and after a ramp response during a GlyR current hump were almost unchanged, and therefore the time course of the humps was not affected by ramp responses. This confirmed that the net Cl− flux across the cell membrane during a ramp response did not significantly alter ECl. See also Supplementary Fig. S1. Bottom traces are the expanded traces of single voltage ramps (upper traces) and superimposed current responses to voltage ramps before and during glycine application (lower traces). Dotted lines indicate the voltage levels at which the superimposed current traces intersected, corresponding to ECl. (B) Plot of ECl in cell groups of Mock (n = 10), WT (n = 12), E50_Q93del & A191V (n = 12), and S323P & M415V (n = 11). *P < 0.03, **P < 0.01 by REGW F-test. (C) Plot of ECl in cells transfected with single vectors encoding WT (n = 11), E50_Q93del (n = 12), A191V (n = 10), S323P (n = 10), and M415V (n = 10). **P < 0.01 by Dunnett’s two-sided t-test.
