(A) Intracellular pH (pHi) of the ligated efferent ductules from WT (n = 9) mice or Gnaq+/- (n = 9) mice was measured by carboxy-SNARF. (B). The whole-cell Cl- current of the IADGRG2-ED elicited by voltage steps between −100 mV and +100 mV in representative ADGRG2 promoter-RFP-labeled efferent ductule cells derived from Gnaq+/- mice, their WT littermates, or WT murine cells incubated with the PKC inhibitor Ro 31–8220 (500 nM). The whole-cell Cl- IADGRG2-ED current was recorded with a CsCl pipette solution (101 mM CsCl, 10 mM EGTA, 10 mM Hepes, 20 mM TEACl, 2 mM MgATP, 2 mM MgCl2, 5.8 mM glucose, pH7.2, with D-mannitol compensated for osm 290) and a bath solution containing 138 mM NaCl, 4.5 mM KCI, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.4 with D-mannitol compensated for osm 310. (C) Corresponding I-V curves of the whole-cell Cl- currents recorded in (B). WT (n = 6), Gnaq+/- (n = 6), WT +Ro 31–8220 (n = 6). (D) Corresponding bar graph of the average current densities (pA/pF) measured at 100 mV according to (C). (E) Co-localization of ADGRG2 (red) and Gq (green) in the male efferent ductules. Scale bars, 50 μm. (F) Co-localization of Gq (red) and acetylated-tubulin (yellow) in the male efferent ductules. Scale bars, 50 μm. (G) IP1 levels in the brain tissues, ligated efferent ductules, and livers of WT (n = 9) or Adgrg2-/Y (n = 9) mice in response to ATP (5 mM) or control vehicles, measured by ELISA. (H) cAMP concentrations in the brains, ligated efferent ductules, and livers of WT (n = 9) or Adgrg2-/Y (n = 9) mice were measured using ELISA. (8A,8D,8G-H) *p<0.05, **p<0.01, ***p<0.001, Adgrg2-/Y mice or Gnaq+/-mice compared with WT mice. #p<0.05, ##p<0.01, ###p<0.001, ATP- or Ro 31–8220-treated cells were compared with control vehicles. n.s., no significant difference. At least three independent biological replicates were performed for Figure 8A,D,G and H.