Fig 2: Effect of NPS-2143 on plasma PTH, calcium, and phosphate of Dsk7 mice at 0, 1, 2, 6, and 24 hours after dose.(A–C) Plasma PTH, (D–F) plasma adjusted-calcium, and (G–I) plasma phosphate concentrations of WT (+/+), Dsk7/+, and Dsk7/Dsk7 mice, respectively. Mean values for the respective groups are indicated by solid bars. n = 4–7 mice per study time point. Squares, males; circles, females. *P < 0.05, **P < 0.01. A Kruskal-Wallis test followed by Dunn’s test for nonparametric pairwise multiple comparisons were used for analysis of A–I.

Fig 3: Intracellular calcium responses of the Val62 Ga11 mutant and effect of NPS-2143 treatment.(A) Fluorescence microscopy of HEK293 cells stably expressing CaSR (HEK-CaSR) and transiently transfected with WT Ile62 or mutant (m) Val62 pBI-CMV2-GNA11 constructs. GFP expression in these cells indicates successful transfection and expression by these constructs. Scale bars: 10 µm. (B) Western blot analysis of lysates from HEK-CaSR cells used for flow cytometry experiments. Transient transfection with WT or mutant Val62 expression constructs resulted in overexpression of Ga11 and GFP. Calnexin, a housekeeping protein, and untransfected cells (Supplemental Figure 2) were used as controls. (C) Ca2+i response to changes in [Ca2+]o of HEK-CaSR cells transfected with WT or Val62 Ga11 mutant. The Ca2+i responses to changes in [Ca2+]o are expressed as a percentage of the maximum normalized responses and shown as the mean ± SEM of 5–8 assays from 2 independent transfections. The Val62 Ga11 mutant led to a leftward shift in the concentration-response curve (red line). The addition of 20 nM NPS-2143 rectified the leftward shift of the Val62 Ga11 mutant (red dashed line), whereas 40 nM NPS-2143 led to a rightward shift of the mutant concentration-response curve (pink dashed line) compared with WT (black line). (D–F) Histograms showing mean ± SEM EC50 values, % maximal signaling responses, and Hill coefficients, respectively, for cells expressing WT (open bar) or Val62 Ga11 mutant (black bar) proteins and for mutant-expressing cells treated with NPS-2143 (patterned bars). **P < 0.01, ***P <0.001. F-test for C–D. Mann-Whitney U test for E–F.

Fig 4: Ca2+i responses of the Gly195 Ga11 mutant and in vitro effect of cinacalcet treatment.(A) Fluorescence microscopy of untransfected (UT) HEK293 cells stably expressing calcium-sensing receptor (CaSR) (HEK-CaSR) and of HEK-CaSR cells transiently transfected with WT (Asp195) or mutant (m) Gly195 pBI-CMV2-GNA11-GFP constructs. GFP expression in these cells indicates successful transfection and expression by these constructs. Scale bar: 10 µm. (B) Western blot analysis of lysates from HEK-CaSR cells used for intracellular calcium (Ca2+i) experiments. Transient transfection with WT or mutant Gly195 G-protein subunit a11 (Ga11) expression constructs resulted in overexpression of Ga11 and GFP, whereas UT cells showed only endogenous Ga11 protein expression. All cells expressed the CaSR. The calnexin and GAPDH proteins were used as loading controls. (C) Ca2+i responses to changes in [Ca2+]o of HEK-CaSR cells expressing WT or Gly195 Ga11 mutant proteins. The Ca2+i responses are expressed as a percentage of the maximum normalized responses and shown as the mean ± SEM of 8 independent transfections. The Gly195 Ga11 mutant led to a rightward shift in the concentration-response curve (red line) compared with cells expressing WT Ga11 (black line). The addition of 10 nM cinacalcet (Cin) normalized the shift of the mutant concentration-response curve (blue line). (D) Histogram showing the mean half-maximal concentration (EC50) with 95% CI of WT cells (black), Gly195 mutant cells (red), and Gly195 mutant cells treated with 10 nM cinacalcet (blue). Statistical analysis was performed using the F-test. ****P < 0.0001 compared with WT.

Fig 5: In vivo effect of cinacalcet on plasma PTH, calcium, and phosphate concentrations of Gna11+/+, Gna11+/195G, and Gna11195G/195G mice.(A–C) Plasma parathyroid hormone (PTH), (D–F) plasma albumin–adjusted calcium, and (G–I) plasma phosphate concentrations are shown at 0, 1, 2, 4, 6, and 24 hours following oral gavage administration of a single 30 mg/kg cinacalcet dose. Mean values for the respective groups are indicated by solid bars. n = 4–5 mice per study time point. Squares, males; circles, females. A Kruskal-Wallis test followed by Dunn’s test for nonparametric pairwise multiple comparisons were used for analysis of A–I. *P < 0.05, **P < 0.01 compared with respective untreated mice. $Untreated Gna11195G/195G mice were significantly (P < 0.05) hypercalcemic compared with untreated Gna11+/+ mice.