Table 1.

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

MutantEC50 (mM)Emax (%)(n)Inhibition by Acidosis (%)Stimulation by Alkalosis (%)(n)
WT-CaR4.3±0.3100±1916−20±214±320
H134V4.3±0.393±196−22±69±49
H192V7.6±1.0a118±165−22±934±137
H254V3.9±0.690±226−10±535±96
H312V3.3±0.580±85−26±532±106
H338V5.7±0.1162±174−22±716±46
H344V6.1±0.5b82±68−38±1055±266
H359V5.2±0.5152±228−24±1311±56
H377V5.8±0.7143±118−43±619±127
H413V3.9±0.4104±205−16±913±39
H429V5.7±0.791±113−13±139±37
H463V5.9±1.190±136−40±523±66
H463V/H466V4.7±0.370±99−17±821±57
H495V4.8±0.6180±523−11±711±68
H766V3.3±0.2131±99−22±420±89
C482S3.7±0.781±393−36±526±87
H429V/H495V5.0±0.453±77−35±419±57
H41V>109−43±622±118
H595V>109−22±713±59
  • 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 pHo 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 CaRH192V and CaRH344V 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 CaRH41V and CaRH595V 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 pHo sensitivity (one-way ANOVA with Dunnett) in response to pathophysiologic alkalosis (pHo 7.6) or acidosis (pHo 7.2) in the presence of 3.5 mM Ca2+o. Because CaRH41V and CaRH595V 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; Emax, maximal responsiveness. n=coverslips, from a minimum of two independent transfections in the case of the pHo experiments.

  • a P<0.05 by one-way ANOVA, Dunnett post hoc test.

  • b P<0.001 by one-way ANOVA, Dunnett post hoc test.