Figure 1.

CLP257 does not affect KCC2 function. (a) Values of intracellular Cl− in NG108-15 cells were unchanged after 5-h exposure to CLP257 (30 μM) (n = 22 cells for DMSO and 18 cells for CLP257; t = 0.57, P = 0.57, unpaired). (b) Cl− imaging demonstrated that CLP257 (30 μM; orange trace) increased intracellular Cl− values relative to those in the control condition (high [Cl−]_o; red trace), while addition of VU0463271 (VU, 15 μM; purple trace) further increased values. VU0463271 applied by itself (blue trace) also elevated intracellular Cl−. Values were normalized to the difference between the high [Cl−]_o andlow [Cl−]_o (black trace) conditions. Each trace represents the mean ± s.e.m. of fluorescence recordings from five independent cultures (Online Methods). The green trace shows expected values based on the original CLP257 report². (c) Immunoprecipitation (IP) and immunoblot (IB) analysis of KCC2 in commercially available NG108–15 cells and NG108-cl cells obtained from Laval University revealed no KCC2 protein expression, in contrast to high expression in rat cortical neurons (Rat ctx). The blot shown is representative of three independent experiments. (d) Tl⁺ influx assays conducted in HEK293 cells exogenously expressing KCC2 showed that CLP257 (50 μM; green traces) did not increase KCC2-mediated Tl⁺ influx as compared to DMSO (black traces), whereas NEM (100 μM; blue traces) increased KCC2 activity and VU0463271 (10 μM; pink traces) decreased it. The red arrow indicates the time of Tl⁺ addition. (e) Values indicating the rate of change in glycine responses measured during [K⁺]o elevation (Cl− uptake over 2 min) or [K⁺]o reduction (Cl− extrusion over 2 min). CLP257 did not increase the rate of uptake (n = 7 cells for DMSO and 6 cells for CLP257; t = 0.28, P = 0.79, unpaired) or the extrusion rate (n = 7 cells for DMSO and 6 cells for CLP257; t = 0.37, P = 0.71, unpaired). (f) CLP257 did not increase KCC2 surface expression. Representative images of total KCC2 signal (F_t), membrane-localized KCC2 signal (F_m) and internalized KCC2 signal (F_i) in N2a cells expressing KCC2-pHext. Cell shape is shown in dark green in each image; scale bar, 15 μm. Images are representative of four independent experiments. (g) CLP257 rapidly and reversibly potentiated whole-cell GABA_A currents activated by a subsaturating concentration of muscimol in cultured hippocampal neurons. (h) Concentration–response relationship in CLP257 potentiation of muscimol- activated currents in cultured neurons (n = 7–14 neurons for each concentration; half-maximal effective concentration (EC50) of CLP257 = 4.9 μM). (i) VU0463271 (10 μM; n = 9 neurons) and shRNA-mediated knockdown of KCC2 (n = 12 neurons) did not prevent potentiation of GABA_A receptor currents by CLP257 (control, untreated), n = 13 neurons; one-way ANOVA: F(2, 31) = 1.11, P = 0.34; Holm–Sidak’s multiple-comparisons test). All data are reported as the mean ± s.e.m.