Figure 3. Intracellular calcium responses of the Val62 Gα₁₁ 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 Gα₁₁ 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 Gα₁₁ 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 Gα₁₁ 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 Gα₁₁ 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 Gα₁₁ 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.