Figure 2. Ca²⁺_i responses of the Gly195 Gα₁₁ 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 α₁₁ (Gα₁₁) expression constructs resulted in overexpression of Gα₁₁ and GFP, whereas UT cells showed only endogenous Gα₁₁ 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 Gα₁₁ 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 Gα₁₁ mutant led to a rightward shift in the concentration-response curve (red line) compared with cells expressing WT Gα₁₁ (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.