Mutation of alternative pH-sensing residue (His167) in GPR4 blunts CO₂-stimulated breathing. A, Sanger sequencing trace from an F1 heterozygote showing targeted alterations in Gpr4 genomic sequence after CRISPR/Cas9 editing, with substitution of Phe for His167 and introduction of silent BspHI and SacI restriction sites. B, CO₂-stimulated V_E (left), V_T (upper right) and fR (lower right) of GPR4(H167F) and WT control (N = 10 and 11, P55–P136) mice in response to increasing levels of inspired CO₂ (balance O₂). P displayed are from two-way RM ANOVA with Šidák's multiple-comparison test: F_(1,19)= 13.03; p = 0.0019 for genotype and F_(4,76)= 10.06; p < 0.0001 for CO₂ exposure × genotype. C, Minute ventilation of H167F and WT control mice during exposure to the indicated levels of inspired O₂. Two-way RM ANOVA analysis with Šidák's multiple-comparison test: F_(1,19)= 0.4037; p = 0.5328 for genotype and F_(2,38)= 1.823; p = 0.1754 for O₂ exposure × genotype.