Table 1.

Characterization of the Ca²⁺_o-sensitivity, relative maximal responses, and pH_o sensitivity of the CaR histidine and free cysteine mutants versus wild-type

Mutant	EC50 (mM)	Emax (%)	(n)	Inhibition by Acidosis (%)	Stimulation by Alkalosis (%)	(n)
WT-CaR	4.3±0.3	100±19	16	−20±2	14±3	20
H134V	4.3±0.3	93±19	6	−22±6	9±4	9
H192V	7.6±1.0a	118±16	5	−22±9	34±13	7
H254V	3.9±0.6	90±22	6	−10±5	35±9	6
H312V	3.3±0.5	80±8	5	−26±5	32±10	6
H338V	5.7±0.1	162±17	4	−22±7	16±4	6
H344V	6.1±0.5b	82±6	8	−38±10	55±26	6
H359V	5.2±0.5	152±22	8	−24±13	11±5	6
H377V	5.8±0.7	143±11	8	−43±6	19±12	7
H413V	3.9±0.4	104±20	5	−16±9	13±3	9
H429V	5.7±0.7	91±11	3	−13±13	9±3	7
H463V	5.9±1.1	90±13	6	−40±5	23±6	6
H463V/H466V	4.7±0.3	70±9	9	−17±8	21±5	7
H495V	4.8±0.6	180±52	3	−11±7	11±6	8
H766V	3.3±0.2	131±9	9	−22±4	20±8	9
C482S	3.7±0.7	81±39	3	−36±5	26±8	7
H429V/H495V	5.0±0.4	53±7	7	−35±4	19±5	7
H41V	>10	–	9	−43±6	22±11	8
H595V	>10	–	9	−22±7	13±5	9

Two independent series of experiments tested (1) the Ca²⁺_o senstivity and maximal response for each CaR mutant (extracellular histidine or free cysteine residues replaced with valine or serine respectively) versus wild-type and (2) the effect of decreasing or increasing pH_o by 0.2 unit on receptor responsiveness. All but four of the CaR mutants exhibited Ca²⁺_o sensitivity not significantly different from wild-type CaR. For CaR^H192V and CaR^H344V the EC₅₀ values for Ca²⁺_o (mM) were increased significantly. Regarding maximal responsiveness to Ca²⁺_o, none of these CaR mutants responded differently to wild-type (Kruskal–Wallis, Dunn multiple comparison test) with the exception of CaR^H41V and CaR^H595V that required additional cotreatment with a positive allosteric modulator (1 mM R-467) to achieve robust Ca²⁺_i mobilization (not shown). Next, none of the mutants exhibited significantly altered CaR pH_o sensitivity (one-way ANOVA with Dunnett) in response to pathophysiologic alkalosis (pH_o 7.6) or acidosis (pH_o 7.2) in the presence of 3.5 mM Ca²⁺_o. Because CaR^H41V and CaR^H595V lacked responsiveness to 3.5 mM Ca²⁺_o, the percentage change values quoted for them are not directly relative to wild-type CaR, but merely represent the percentage change of their CaR response from the immediately prior response in pH 7.4. EC₅₀ half maximal effective concentration; E_max, maximal responsiveness. n=coverslips, from a minimum of two independent transfections in the case of the pH_o experiments.

^aP<0.05 by one-way ANOVA, Dunnett post hoc test.

^bP<0.001 by one-way ANOVA, Dunnett post hoc test.