Table 1. Regression results from fitted GABA-evoked responses at wild-type and concatenated GABAARs.
GABA | ||||||
---|---|---|---|---|---|---|
Construct | Subunit | Emax | EC50 (µM) | pEC50 | n | GABAmax (nA) |
α1 + β2 1:50 | 0.99 ± 0.01 | 2.6 | 5.58 ± 0.03 | 12 | 1,500 ± 200 | |
α1 + β2 50:1 | 0.99 ± 0.02 | 3.2 | 5.49 ± 0.03 | 17 | 750 ± 120 | |
α1 + β2 + γ2 5:1:5 | 1.00 ± 0.01 | 22 | 4.66 ± 0.03 | 10 | 3,200 ± 400 | |
Dimeric constructs | ||||||
β2-23-α1 | 1.00 ± 0.02 | 3.0 | 5.53 ± 0.04 | 12 | 480 ± 100 | |
β2-23-α1 | β2 | 1.00 ± 0.02 | 2.6 | 5.58 ± 0.03 | 10 | 1,900 ± 200 |
β2-23-α1 | γ2 | 1.00 ± 0.02 | 9.2 | 5.04 ± 0.04 | 8 | 8,400 ± 600 |
α1-10-β2 | 1.00 ± 0.01 | 2.8 | 5.55 ± 0.03 | 11 | 820 ± 190 | |
α1-10-β2 | β2 | 1.00 ± 0.01 | 1.8 | 5.75 ± 0.02 | 13 | 1,800 ± 200 |
α1-10-β2 | γ2 | 1.00 ± 0.02 | 14 | 4.84 ± 0.04 | 10 | 8,100 ± 600 |
α1-9a-β2 | 1.00 ± 0.01 | 3.2 | 5.49 ± 0.03 | 10 | 190 ± 60 | |
α1-9a-β2 | β2 | 1.00 ± 0.02 | 1.7 | 5.77 ± 0.04 | 11 | 1,200 ± 300 |
α1-9a-β2 | γ2 | 0.99 ± 0.01 | 17 | 4.77 ± 0.03 | 20 | 11,000 ± 1,000 |
α1-6a-β2 | 0.99 ± 0.01 | 5.7 | 5.24 ± 0.03 | 11 | 140 ± 30 | |
α1-6a-β2 | β2 | 1.00 ± 0.01 | 2.3 | 5.65 ± 0.02 | 11 | 1,300 ± 300 |
α1-6a-β2 | γ2 | 1.00 ± 0.02 | 41 | 4.38 ± 0.04 | 10 | 3,100 ± 300 |
α1-3a-β2 | 1.00 ± 0.02 | 10 | 4.99 ± 0.04 | 10 | 89 ± 28 | |
α1-3a-β2 | β2 | 1.00 ± 0.02 | 3.2 | 5.49 ± 0.03 | 9 | 630 ± 110 |
α1-3a-β2 | γ2 | 0.99 ± 0.02 | 34 | 4.46 ± 0.04 | 12 | 4,300 ± 500 |
α1-0a-β2 | N/A | 19 | 34 ± 10 | |||
α1-0a-β2 | β2 | 1.00 ± 0.01 | 3.5 | 5.45 ± 0.02 | 14 | 630 ± 100 |
α1-0a-β2 | γ2 | 0.99 ± 0.03 | 130 | 3.88 ± 0.05 | 11 | 500 ± 140 |
α1-(-1a)-β2 | N/A | 21 | No current | |||
α1-(-1a)-β2 | β2 | 0.99 ± 0.01 | 2.6 | 5.58 ± 0.03 | 7 | 400 ± 140 |
α1-(-1a)-β2 | γ2 | 0.99 ± 0.02a | 140a | 3.84 ± 0.04a | 18 | 7.8 ± 1.9b |
α1-(-2a)-β2 | N/A | 15 | No current | |||
α1-(-2a)-β2 | β2 | 1.00 ± 0.01 | 2.6 | 5.59 ± 0.03 | 11 | 470 ± 90 |
α1-(-2a)-β2 | γ2 | 1.00 ± 0.01a | 130a | 3.90 ± 0.03a | 17 | 8.0 ± 3.6b |
α1-(-3a)-β2 | N/A | 21 | No current | |||
α1-(-3a)-β2 | β2 | 0.96 ± 0.04 | 4.0 | 5.40 ± 0.08 | 8 | 40 ± 9 |
α1-(-3a)-β2 | γ2 | 1.00 ± 0.01a | 64a | 4.20 ± 0.02a | 24 | 2.3 ± 0.9b |
Tetrameric constructs | ||||||
α1-10-β2-α1-β2 | N/A | 13 | 60 ± 13 | |||
α1-10-β2-α1-β2 | γ2 | 0.99 ± 0.02 | 32 | 4.50 ± 0.03 | 17 | 6,500 ± 1,000 |
α1-10-β2-α1-γ2 | N/A | 15 | 150 ± 25 | |||
α1-10-β2-α1-γ2 | β2 | 0.96 ± 0.03 | 34 | 4.47 ± 0.05 | 19 | 4,700 ± 400 |
Pentameric constructs | ||||||
α1-(-1a)-β2-α1-β2-γ2 | 0.99 ± 0.01 | 390 | 3.41 ± 0.02 | 19 | 320 ± 40 | |
α1-(-1a)-β2-α1-γ2-β2 | 1.00 ± 0.01 | 330 | 3.49 ± 0.02 | 16 | 3,500 ± 400 | |
γ2-15a-β2-α1-β2-α1 | 0.99 ± 0.03 | 51 | 4.30 ± 0.04 | 11 | 4,900 ± 600 | |
γ2-13a-β2-α1-β2-α1 | 0.99 ± 0.02 | 70 | 4.16 ± 0.03 | 12 | 5,300 ± 500 | |
γ2-11a-β2-α1-β2-α1 | 0.99 ± 0.01 | 110 | 3.96 ± 0.02 | 17 | 3,300 ± 500 | |
γ2-10a-β2-α1-β2-α1 | 0.99 ± 0.02 | 120 | 3.92 ± 0.04 | 10 | 3,100 ± 700 | |
γ2-9a-β2-α1-β2-α1 | 1.00 ± 0.01 | 120 | 3.92 ± 0.02 | 15 | 890 ± 160 | |
Mutant constructs | ||||||
α1-9a-β2 | γ2(A118R) | 0.99 ± 0.02 | 30 | 4.52 ± 0.04 | 13 | 8,700 ± 1,500 |
α1-0a-β2 | γ2(A118R) | 1.00 ± 0.01 | 110 | 3.98 ± 0.02 | 9 | 600 ± 90 |
α1R94A + β2 + γ2 5:1:5 | 0.99 ± 0.02 | 1,100c | 2.97 ± 0.03c | 10 | 7,100 ± 400 | |
γ2-11a-β2-α1-β2-α1R94A | 1.01 ± 0.01 | 2,600c | 2.58 ± 0.02c | 14 | 3,600 ± 300 | |
γ2-11a-β2-α1-β2-α1R94A | α1 | 0.97 ± 0.02c,d | 2,000c,d | 2.70 ± 0.03c,d | 12 | 1,700 ± 200 |
Data points were fitted to the Hill equation with the bottom set to 0 and Hill slope set to 1 by nonlinear regression. Normalized fitted maximal responses of GABA are presented as Emax ± SEM. Fitted potencies of GABA are presented as EC50 in µM and pEC50 ± SEM where p = −Log for the indicated number (n) of individual oocytes. The average maximal current obtained with GABAmax applications is presented as GABAmax ± SEM in nA for the tested oocytes. For α-10-β+γ2, a GABA concentration of 3,160 µM was used to establish GABAmax-evoked peak currents amplitudes, whereas a concentration of 316 µM was used for all remaining cRNA combinations of wild-type free subunits, binary constructs, and tetrameric constructs. For pentameric constructs and cRNA combinations with mutant subunits, the GABAmax concentration was adjusted according to the EC50 value of GABA CRRs: for CRRs with EC50 values <70 µM, a concentration of 316 µM was used; for CRRs with EC50 values between 70 and 500 µM, a concentration of 3,160 or 10,000 µM was used; and for CRRs with EC50 values >500 µM, a concentration of 10,000 µM was used. N/A, not applicable; No current, no GABAmax-evoked peak-current amplitude exceeded 5 nA in any of the tested oocytes.
Data were from n = 5 experiments performed on oocytes that had been incubated for 5–6 d following cRNA injection and selected based on GABAmax-evoked current amplitudes; note that data for these n = 5 oocytes are not included in the number of oocytes tested or GABAmax results.
Given the GABA EC50 value at this construct combination, the utilized 316-µM GABAmax concentration represents a submaximal EC70–80 concentration; an EC100 concentration would thus be expected to give values that are 20–30% larger.
Only 80–90% of the fitted curve is covered by data points (Fig. 9 C) and derived fitted values could therefore be seen as lower limits.
These values are derived from first-order fitting; however, data are statistically better represented by second-order fitting as described in the text.