Fig. 2. Bicarbonate ions activate GPR30 signalling through the G_q family of G proteins.

a TGFα shedding assay using HEK293 cells devoid of G_αq/11/12/13 and transfected with the indicated subtypes of G_α proteins. Blue and magenta bars indicate G_αi and G_αq family candidates, respectively. b, c cAMP assay using HEK293 cells transiently (b) and stably (c) expressing hGPR30. Sodium bicarbonate did not inhibit forskolin (FSK)-dependent cAMP production in either cell line. d Calcium mobilisation assay using Fluo-8-loaded HEK-GPR30. The cells were treated with vehicle, 3.3 mM, and 11 mM NaHCO₃. Bicarbonate-induced intracellular calcium increase was completely abolished by the G_αq/11/14 inhibitor YM-254890 (YM, 1 µM, 45 min) but not by pertussis toxin (PTX, 100 ng/ml, 16 h). e, f HEK293 cells transiently expressing hGPR30 were pretreated with YM-254890 (e) or PTX (f) and then treated with 11 mM NaHCO₃ for the indicated periods. β-actin served as the loading control. g GPR30-dependent phosphorylation of ERK1/2 was evaluated using western blotting. HEK293 cells transiently expressing hGPR30 were treated with vehicle, 11 mM NaHCO₃, or 100 nM E₂ and harvested at 15 and 30 min after stimulation. β-actin serves as a loading control. h, i GPR30-dependent accumulation of inositol phosphates. HEK293 cells transiently expressing hGPR30 (h) and MCF-mGPR30 cells (i) were treated with indicated sodium bicarbonate concentrations. Nonlinear regression (four parameters) was performed. The EC₅₀ values of sodium bicarbonate were 11.40 mM (h) and 12.20 mM (i). Statistical analysis: two-tailed unpaired t-test with Bonferroni’s correction after two-way ANOVA (a–d). Data are presented as mean values ± SEM. P values are shown if significant. ns indicates no significant difference. Source data are provided as a Source Data file.