Article Figures & Data
Figures
- Fig. 1.
Alignment of loop C domains from different LGICs. The β2 loop C domain of the GABA binding site is aligned with homologous domains from the benzodiazepine binding site of the GABAA α1 subunit, the acetylcholine binding site of the nicotinic acetylcholine receptor α1 subunit, and the glycine binding site of the glycine receptor α1subunit. Residues that have been predicted to be in or near the binding pocket by photo-affinity labeling or mutagenesis are shown inbold (Galzi and Changeux, 1994). Residues in β2 that were mutated to cysteines are denoted by aCabove the wild-type residue.
- Fig. 2.
GABA dose–response curves and P4S currents.A, GABA dose–response relationships for wild-type α1β2 receptors (●) and three representative mutants: α1β2-R207C (▴), α1β2-S201C (♦), and α1β2-Y205C (▾). Data were fit by nonlinear regression as described in Materials and Methods. All data points are normalized to Imax-GABA and are shown as mean responses ± SEM from four or more cells.B, Current traces recorded from oocytes expressing wild type or α1β2-S201C. Arrowsindicate a 5 sec application of saturating P4S (wild type, 1 mm; S201C, 10 mm) or GABA (wild type, 1 mm; S201C, 100 mm). Line breakin current trace represents 5 min wash with ND96. C, Bar graph denoting P4S efficacy of wild-type and mutant receptors asImax-P4S/Imax-GABAwhere values given as mean ± SEM follow: α1β2, 0.50 ± 0.03,n = 4; α1β2-F200C, 0.45 ± 0.06, n = 4; α1β2-S201C, 0.12 ± 0.01,n = 3; and α1β2-R207C, 0.21 ± 0.02, n = 4. * p< 0.01 indicates values that are significantly different from wild type calculated using a one-way ANOVA with a Dunnett's post hoc test.
- Fig. 3.
Effects of MTSEA-biotin on wild-type and mutant GABAA receptors. A, Representative current traces demonstrating the effect of MTSEA-biotin treatment (2 mm, 2 min) on currents from wild-type and Y205C-containing receptors. For wild-type traces, [GABA] is 3 μm, and for Y205C traces, [GABA] is 30 mm. B, Effect of MTSEA-biotin treatment on all mutants shown as % change = ([IGABA-post MTSEA-biotin/IGABA-pre MTSEA-biotin] − 1) × 100. Results represent the mean ± SEM of at least three experiments. Black bars indicate that the percent change is significantly different from wild type (p < 0.01). Gray barsindicate no significant difference from wild type (p > 0.05).
- Fig. 4.
Measurement of MTSEA-biotin reaction rates.A, B, Examples of traces recorded during experiments measuring the reaction rate of MTSEA-biotin with α1β2-R207C receptors. Downward deflections represent inward current elicited by a 5 sec application of 300 μm GABA (≈EC50).Arrows indicate either 10 sec application of 200 μm MTSEA-biotin (A) or a 20 sec coapplication of MTSEA-biotin plus 1 μm SR-95531 (B). C, NormalizedIGABA plotted as a function of cumulative time of MTSEA-biotin exposure. Single exponential curve fits illustrate the effect of various compounds on the reaction rate of MTSEA-biotin with α1β2-R207C receptors. Data points are normalized to the current measured at t = 0 and are presented as mean ± SEM. PB, Pentobarbital.
- Fig. 5.
Summary of effect of GABA, SR-95531, and pentobarbital on the rate at which MTSEA-biotin modifies introduced cysteines. Second-order rate constants were calculated for each reaction, and for each mutant, the rates were normalized to the control rate (rate measured when no other compound is present). *p < 0.01 indicates that rate is significantly different from control rate. All data represent the mean ± SEM of at least three experiments.
- Fig. 6.
Graphic summary of results. Barsindicate which residues fall into each of the following categories:Mediates KD-GABA, mutation of this residue alters microscopic affinity for GABA; Mediates Efficacy, mutation of this residue reduces efficacy of P4S;Accessible to MTS, we can detect reaction of MTSEA-biotin with a cysteine introduced at this position; In Binding Pocket, the rate at which MTSEA-biotin reacts with a cysteine introduced at this residue is slowed by the presence of both GABA and SR-95531. Relative Rxn. Rate, The height of thebar is scaled to the log ofk2 for each mutant with MTSEA-biotin.
Tables
- Table 1.
Apparent affinites of wild-type and mutant receptors for GABA, SR-95531, and pentobarbital
Receptor GABA SR-95531 PB EC50(μm) nH n wt/mut Ki(μm) n wt/mut EC50(μm) n wt/mut α1β2 Wild type 4.3 ± 1.2 1.1 8 1.0 0.12 ± 0.02 8 1.0 570 ± 66 5 1.0 α1β2-V199C 3.1 ± 0.4 1.4 4 0.7 0.08 ± 0.01 3 0.6 577 ± 195 5 1.0 α1β2-F200C 1292 ± 170* 1.1 4 300 4.34 ± 2.6* 3 36.2 712 ± 96 3 1.2 α1β2-S201C 725 ± 160* 0.5* 4 170 1.42 ± 0.3* 3 11.8 1038 ± 425 3 1.8 α1β2-T202C 61100 ± 10900* 1.0 5 14000 1.90 ± 0.4* 3 15.8 690 ± 170 3 1.2 α1β2-G203C 20480 ± 6990* 1.0 10 4800 4.67 ± 0.4* 4 38.9 1088 ± 70 3 1.9 α1β2-S204C 1.5 ± 0.3 1.1 6 0.3 0.05 ± 0.03 5 0.4 427 ± 86 5 0.7 α1β2Y205C 78000 ± 7070* 1.4 5 18000 12.40 ± 1.5* 3 103.3 800 ± 42 4 1.4 α1β2-P206C 2.6 ± 1.0 1.2 4 0.6 0.53 ± 0.13 6 4.4 347 ± 46 4 0.6 α1β2-R207C 310 ± 30* 1.3 4 70 0.11 ± 0.02 4 0.9 480 ± 72 4 0.8 α1β2-L208C 2.7 ± 0.5 1.5 5 0.6 0.07 ± 0.02 3 0.6 327 ± 77 5 0.6 α1β2-S209C 5.4 ± 1.1 1.1 3 1.3 0.11 ± 0.01 3 0.9 583 ± 105 3 1.0 EC50 and Ki values are presented as mean ± SEM. An asterisk indicates that the value is significantly different from wild type (p < 0.01). PB, Pentobarbital; wt, wild type; mut, mutant.
- Table 2.
Summary of second-order rate constants for reaction of MTSEA-biotin with introduced sufhydryls
Receptor Control GABA SR-95531 1 mm PB 50 μm PB k2(M−1s−1) n k2(M−1s−1) n k2(M−1s−1) n k2(M−1s−1) n k2(M−1s−1) n α1β2-G203C 257,600 ± 32,700 3 184,400 ± 14,300 3 317,700 ± 19,500 3 579,200 ± 9900* 3 400,100 ± 7300* 3 α1β2-S204C 12,060 ± 980 5 6050 ± 980* 5 3735 ± 310* 3 5656 ± 1180* 3 14,990 ± 3300 3 α1β2-Y205C 955 ± 150 5 410 ± 15* 4 285 ± 5* 3 870 ± 40 3 ND 0 α1β2-P206C 276 ± 41 4 715 ± 70* 3 398 ± 16 3 385 ± 25 3 352 ± 17 3 α1β2-R207C 585 ± 40 6 100 ± 9* 4 135 ± 10* 3 370 ± 32* 3 690 ± 65 3 α1β2-S209C 120 ± 2 4 67 ± 11* 4 40 ± 7* 3 77 ± 8* 3 125 ± 3 3 Second-order rate constants (k2) were calculated by dividing psuedo-first-order rate constants (k1; see Materials and Methods) by the concentration of MTSEA-biotin used during rate experiments, which were as follows: G203C, 1 μm; S204C, 10 μm; Y205C, P206C, and R207C, 200 μm; and S209C, 1 mm. Concentrations of GABA and SR-95531 present during the MTSEA-biotin reaction, which varied according to the affinity of the mutant receptor for each compound, were always between EC60–IC80 for GABA and IC90–IC95 for SR-95531. An asterisk indicates that the rate is significantly different from control (p < 0.01). PB, Pentobarbital; ND, not determined. Values are mean ± SEM.
