Fig. 4: Diffusion of CO2 across membrane controls pHi homeostasis.
From: Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm
a Scheme of the difference between an open experimental system in equilibrium with air (pCO2 = 0.3 mmHg) and a quasi-closed system in equilibrium with body fluid (pCO2 = 40 mmHg). b Time course of alkalization after a switch from pHo 6.5 to pHo 7.7 (control, black, same recording as in Fig. 3I) and from pHo 6.5 to pHo 7.5 in the presence of CO2/HCO3− (5%/3 mM, 5%/30 mM) (blue). c Final pHi after incubating sperm for 30 min at the indicated pHo in the absence (grey) or presence of CO2/HCO3− (pHo 6.5 + 5%/3 mM or pHo 7.5 + 5%/30 mM; blue). Two-factor ANOVA followed by post-hoc Tukey’s test for multiple comparison: pHo 6.5 vs. pHo 7.7 p < 0.0001; pHo 6.5 vs. pHo 6.5 CO2/HCO3− p = 0.003; pHo 6.5 vs. pHo 7.5 CO2/HCO3− p = 0.014; pHo 7.7 vs. pHo 6.5 CO2/HCO3− p < 0.0001; pHo 7.7 vs. pHo 7.5 CO2/HCO3− p = 0.23; pHo 6.5 CO2/HCO3− vs. pHo 7.7 CO2/HCO3− p < 0.0001. No significant interaction between the factors (p = 0.09). d Half-time t1/2 of ∆pHi upon a switch from pHo 6.5 to pHo 7.7 without (black) and from pHo 6.5 to pHo 7.5 with CO2/HCO3− (5%/3 mM and 5%/30 mM) (blue; nexp = 3 and 5, respectively). Mann-Whitney test: Ctrl vs. CO2/HCO3− p = 0.08. e Normalized changes in pHrodo Red fluorescence upon switching from pHo 7.35 to pHo 7.35/CO2/HCO3− (5%/25 mM) solution in sperm (black), HEK293 cells (blue), and CHO cells (red). Green trace mixing kinetics in the recording chamber measured with Alexa488. f Extended time scale of panel (e). g HCO3−-induced ∆pHi upon switching from pHo 7.35 to pHo 7.35/CO2/HCO3− (5%/25 mM) in the presence of inhibitors. Hv1, Zn2+ (100 μM); CFTR, CFTRinh172 (100 μM); SLC4, DIDS (100 μM); and SLC9A, amiloride (500 μM; nexp = 10, 11, 3, 4, and 4, respectively). Kruskal–Wallis test followed by post-hoc Dunn’s test for multiple comparison: Ctrl vs. Zn2+, p = 0.7; Ctrl vs. CFTRinh172 p > 0.9; Ctrl vs. DIDS p > 0.9; Ctrl vs. amiloride p = 0.6. h Time constants τ of ∆pHi upon switching from pHo 7.35 to pHo 7.35/CO2 /HCO3− in the absence (grey) or presence of ACZ (100 μM, red) in human sperm, CHO, and HEK293 cells (nexp = 10, 9, 5, 3, 4, and 2, in presented order). Two-factor ANOVA followed by post-hoc Tukey’s test for multiple comparison: sperm vs. sperm ACZ p < 0.0001; sperm vs. CHO p < 0.0001; sperm vs. HEK p = 0.2; CHO vs. HEK p < 0.0001; CHO vs. CHO ACZ p = 0.21; HEK vs. HEK ACZ p < 0.0001; sperm ACZ vs. CHO ACZ p < 0.0001; sperm ACZ vs. HEK ACZ p < 0.0001; CHO ACZ vs. HEK ACZ p = 0.009. Significant interaction between the factors (p < 0.0001). Box plots represent 25%, 50% (median), and 75% quartiles, with whiskers displaying minimum and maximum data points. Source data is provided as a source data file.
