Table 3

Effect of Ca²⁺ on the affinities and potencies of GABA and baclofen on wild-type and mutant GABA_BR1a receptors

	Ca²⁺ [EC₅₀(μM)]		EGTA [EC₅₀ (μM)]		Ca²⁺Ratio	EGTA Ratio
	GABA	Baclofen	GABA	Baclofen	Ca²⁺Ratio	EGTA Ratio
[¹²⁵I]CGP64213 binding
WT	20.9 ± 0.8	30.2 ± 1.7	780 ± 143	34.2 ± 7.2	0.69	22.8
S247A	5.32 ± 0.44	11.3 ± 0.9	287 ± 45	14.9 ± 11.5	0.42	19.3
S268A	27.8 ± 1.9	43.0 ± 4.1	327 ± 51	28.7 ± 4.7	0.64	11.4
S269A	238 ± 28	28.8 ± 5.4	227 ± 55	31.2 ± 0.9	8.26	7.27
IP formation
WT	2.95 ± 0.20	3.15 ± 0.44	179 ± 9	22.9 ± 2.1	0.93	7.81
S247A	0.44 ± 0.04	0.38 ± 0.04	32.7 ± 11	2.57 ± 0.08	1.16	12.7
S268A	1.02 ± 0.15	0.91 ± 0.41	47.2 ± 0.2	6.22 ± 3.77	1.12	7.58
S269A	14.9 ± 0.8	1.78 ± 0.22	212 ± 13	21.2 ± 1.5	8.37	10.0

[¹²⁵I]CGP64213 binding on membranes prepared from HEK 293 cells expressing the indicated GABA_BR1 receptor was measured in the absence and presence of increasing concentration of the indicated compounds. IC₅₀ values (micromolar) were determined as described under Experimental Procedures. The potencies of GABA and baclofen in activating the wild-type and mutants GABA_Breceptors were determined in HEK 293 cells coexpressing the wild-type and mutant GABA_BR1, the wild-type GABA_BR2, and the chimeric G protein Gqi9. Experiments were conducted in the presence of Ca²⁺, or in the presence of 1 mM EGTA with no added Ca²⁺ (binding experiments), or 5 mM EGTA and 0.1 mM Ca²⁺ (IP experiments). Values are means ± S.E.M. of at least three independent experiments performed in triplicate. In the last two columns, the ratios GABA/baclofen EC₅₀ values measured in the presence (Ca²⁺) or absence (EGTA) of Ca²⁺ are presented.

WT, wild-type.