Table 1. Regression results from fitted... | Rockefeller University Press

Table 1.

Regression results from fitted GABA-evoked responses at wild-type and concatenated GABA_ARs

		GABA
Construct	Subunit	E_max	EC₅₀ (µM)	pEC₅₀	n	GABA_max (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 ± 0.02^{^a}	140^a	3.84 ± 0.04^{^a}	18	7.8 ± 1.9^{^b}
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 ± 0.01^{^a}	130^{^a}	3.90 ± 0.03^{^a}	17	8.0 ± 3.6^{^b}
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 ± 0.01^{^a}	64^{^a}	4.20 ± 0.02^{^a}	24	2.3 ± 0.9^{^b}
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
α1^R94A + β2 + γ2 5:1:5		0.99 ± 0.02	1,100^{^c}	2.97 ± 0.03^{^c}	10	7,100 ± 400
γ2-11a-β2-α1-β2-α1^R94A		1.01 ± 0.01	2,600^{^c}	2.58 ± 0.02^{^c}	14	3,600 ± 300
γ2-11a-β2-α1-β2-α1^R94A	α1	0.97 ± 0.02^c,d	2,000^{^c}^,^{^d}	2.70 ± 0.03^{^c}^,^{^d}	12	1,700 ± 200

		GABA
Construct	Subunit	E_max	EC₅₀ (µM)	pEC₅₀	n	GABA_max (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 ± 0.02^{^a}	140^a	3.84 ± 0.04^{^a}	18	7.8 ± 1.9^{^b}
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 ± 0.01^{^a}	130^{^a}	3.90 ± 0.03^{^a}	17	8.0 ± 3.6^{^b}
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 ± 0.01^{^a}	64^{^a}	4.20 ± 0.02^{^a}	24	2.3 ± 0.9^{^b}
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
α1^R94A + β2 + γ2 5:1:5		0.99 ± 0.02	1,100^{^c}	2.97 ± 0.03^{^c}	10	7,100 ± 400
γ2-11a-β2-α1-β2-α1^R94A		1.01 ± 0.01	2,600^{^c}	2.58 ± 0.02^{^c}	14	3,600 ± 300
γ2-11a-β2-α1-β2-α1^R94A	α1	0.97 ± 0.02^c,d	2,000^{^c}^,^{^d}	2.70 ± 0.03^{^c}^,^{^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 E_max ± 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 GABA_max applications is presented as GABA_max ± SEM in nA for the tested oocytes. For α-10-β+γ2, a GABA concentration of 3,160 µM was used to establish GABA_max-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 GABA_max 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 GABA_max-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 GABA_max-evoked current amplitudes; note that data for these n = 5 oocytes are not included in the number of oocytes tested or GABA_max results.

Given the GABA EC₅₀ value at this construct combination, the utilized 316-µM GABA_max concentration represents a submaximal EC_70–80 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.

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