Estimation of open probability using rate of MK-801 channel block
Data are from whole-cell patch-clamp recordings of GluN2 subunits coexpressed with GluN1 in HEK293 cells. Cells were voltage-clamped at −60 mV, and ligands were applied using rapid solution exchange. Steady-state responses to 1 mM glutamate plus 100 µM glycine at pH 8.0 were inhibited by 5 µM MK-801 and the inhibition time course was determined using a mono-exponential fit. The inhibition time constant (τMK-801) was then used to calculate the rate of inhibition (1/τMK-801), which is linearly correlated to open probability (Popen) assuming MK-801 affinity is constant, using Popen(chimera) = Popen(WT) * (τMK-801(WT)/τMK-801(chimera)). Chimera data were compared with Popen for wild-type GluN1/GluN2A. Data are presented as the mean ± S.E.M.
| GluN2 Subunit | Inhibition τMK-801 | Rate of Block 1/τMK-801 | Cells | Estimated Popen | Fold Change in Popen (Relative to Wild Type) |
|---|---|---|---|---|---|
| ms | s−1 | n | |||
| GluN2A | 71 ± 9 | 15 ± 2 | 6 | 0.48a | — |
| 2A-(2D ATD) | 374 ± 98 | 3.3 ± 0.6 | 6 | 0.10 | 0.22 |
| 2A-(2D S1) | 199 ± 37 | 5.8 ± 1.0 | 5 | 0.18 | 0.38 |
| 2A-(2D S2) | 266 ± 25 | 3.2 ± 0.4 | 5 | 0.12 | 0.26 |
| 2A-(2D S1+S2) | 308 ± 46 | 3.5 ± 0.3 | 7 | 0.11 | 0.23 |
| GluN2D | 1043 ± 98 | 1.0 ± 0.1 | 6 | 0.012a | — |
↵a Indicates previously published Popen values for wild-type receptors determined using single-channel recordings (Yuan et al., 2009). To allow comparison with these values, the inhibition data were generated using the same compositions of intracellular and extracellular solutions as the previous study (i.e., pH 8.0 and 0.5 mM Ca2+ in the extracellular solution). The rate of MK-801 inhibition at pH 8.0 was lower than previously described rates at pH 7.6, consistent with MK-801 having a pKa of 8.37 (i.e., less protonated MK-801 at higher pH) (Dravid et al., 2007). All chimeras were blocked > 95% by 5 µM MK-801.