(A) Bar graphs show the amplitude of GluK1 currents (mean ± SEM) from outside-out patches pulled from wild type and transfected CA1 neurons with indicated plasmids and exposed to 1 or 100 ms applications of 10 mM glutamate and 100 μM GYKI53655 (WT, n=7, 8.57 ± 2.51 pA, *** p < 0.0005; GluK1: n=10, 81.65 ± 11.26 pA; GluK1/Neto1: n=9, 1231.94 ± 242.92 pA, *** p < 0.0001; GluK1/Neto1S3Y/A: n=7, 967.14 ± 138.30 pA, *** p < 0.0005; GluK1/Neto2: n=10, 1022.84 ± 241.81 pA, *** p < 0.0005; GluK1/Neto2S4T/A: n=9, 1035.22 ± 115.00 pA, *** p < 0.0001). All the statistical analyses are compared to GluK1 single overexpression using Mann-Whitney U-test. Sample traces and scale bar are shown to the right. (B) DIV 10 neurons were transfected with HA-GluK1 and Neto1 or Neto2, as indicated. At DIV 13, cells were stained for surface GluK1 and the intensity of surface GluK1 was quantitated (3 dendrites per neuron) using Metamorph analysis software. Scale bar, 20 μm. Images at the bottom of each panel are higher magnification from the enclosed region. Scale bar, 5 μm. (C) Bar graph shows the surface expression of GluK1 (mean ± SEM) from three independent experiments (GluK1: n=34; GluK1/Neto1: n=33; GluK1/Neto2: n=34). An unpaired two-tailed t-test was used to determine the significance of the data: *** p < 0.0001. (D and E) Bar graphs show mean ± SEM GluK1 deactivation (d, GluK1: n=10, 3.7 ± 0.35 ms; GluK1/Neto1: n=7, 3.43 ± 0.42 ms, p > 0.05; GluK1/Neto2: n=10, 5.33 ± 0.46 ms, * p < 0.05) and desensitization (e, GluK1: n=6, 12.42 ± 2.26 ms; GluK1/Neto1: n=8, 4.93 ± 0.59 ms, * p < 0.05; GluK1/Neto2: n=7, 27.49 ± 3.26 ms, ** p < 0.005) from outside-out patches pulled from indicated transfection CA1 neurons and exposed to 1 or 100 ms applications of 10 mM glutamate and 100 μM GYKI53655, respectively. All the statistical analyses are compared to GluK1 single overexpression using Mann-Whitney U-test. Sample traces are shown to the right and are peak-normalized.