Figure 7.
Accessibility pattern of cysteine-substituted mutation at the N-site residue of the M2 segments of NR1 and NR3 after widening the constriction at the outer vestibule of the channel. A, Representative recordings showing modulation by external MTS reagents of NR1(T+2G, N616C)/NR3A mutant channels (designated NR1TG/N616C+3A in the figure for brevity). The smaller MTSEA persistently modified the channel. MTSEA modification could be reduced by preexposure to the pore blocker NMDG (96 mm) (gray tracing at top right). B, NMDG protection assay to test whether a specific amino acid residue lines the channel pore region. Left, Degrees of residual current (=IPost-drugs/IControl × 100%) at 30, 60, 90, and 120 s is plotted to show the temporal delay in action of 0.5 mm MTSEA by the pore blocker NMDG (96 mm). Right, Residual current after MTSEA modification of various combinations of mutants. The first bar indicates lack of modification by MTSEA (96.2 ± 2.2%) of NR1(T+2G)/NR3A mutants. The second bar shows persistent modification of NR1(T+2G, N616C)/NR3A channels by MTSEA (59.8 ± 2.6%). The third to fifth bars indicate that replacement of endogenous cysteines in the permeation pathway with alanines did not affect modification of the NR1(N616C) residue by MTSEA. The last bar illustrates protection from MTSEA modification by preexposure to NMDG (81.3 ± 4.7%). *Statistically significant modification compared with NR1(T+2G)/NR3A channels by an unpaired Student t test. **Significant protection from MTSEA modification on NR1(T+2G, N616C)/NR3A mutant channels by NMDG. Error bars indicate SEM. C, Representative recordings of persistent modulation by external MTSEA of currents from NR1(T+2G)/NR3A(G729C) (left trace). Right, NMDG protection assay. Degree of modulation at 30, 60, 90, and 120 s by MTSEA of NR1(T+2G)/NR3A(G729C) channels is plotted as in B; NMDG had no effect on the kinetics of MTSEA modification. D, Persistent modification of currents from NR1(T+2G)/NR3A(G729C) by external MTSEA could be eliminated by replacement of all three endogenous cysteines in the permeation pathway with alanines (designated as NR1TG+NR3A(3CA)GC) (left trace). Right, Residual current after persistent MTSEA modification of various combinations of mutants. The first bar shows lack of modification by MTSEA (94.1 ± 2.3%) on the NR1/3A(G729C), designated NR1/NR3A(GC) for brevity. The second bar shows modification of NR1(T+2G)/NR3A(G729C) channels by MTSEA (18.8 ± 2.4%). The NR3A(C723A, G729C) mutant, replacing the endogenous cysteine at residue 723 in the M2 segment, did not form function channels with NR1(T+2G). NE, No expression. The fourth and fifth bars indicate that replacement of the two endogenous cysteines in the permeation pathway formed by the M3 segment with alanines (NR3A C753A and C756A) did not affect modification on the NR3A(G729C) residue. The sixth bar shows that replacement of all three endogenous cysteines in the M2 and M3 segments abrogated modification by MTSEA. The seventh bar shows that preexposure to NMDG did not protect NR1(T+2G)/NR3A(G729C) from MTSEA modification. The last bar indicates that widening the outer vestibule of the channel by mutating NR3A(T+2G) also permitted modification of the NR3A(G729C) residue by MTSEA (28.6 ± 1.9%). *Statistically significant modification compared with NR1/NR3A(G729C) channels by an unpaired Student t test. Calibration: 100 nA, 30 s. The number in each column represents oocytes tested.