Solutions

Chemical compounds were disolved in HBSS containing 10 mM HEPES. Chemical compounds used in this study were saccharin (10 mM), SC45647 (0.3 mM), aspartame (10 mM), and sodium cyclamate (30 mM) as sweet taste stimuli and isoproterenol (10 μM) as positive control. We used citric acid (pK_a: 3.09) (concentration 3 mM in HBSS containing 10 mM HEPES, pH 7.4, 6.25, 6.0, 5.75, 5.5, 5.25, 5.0 adjusted using NaOH), and HCl (pKa: −8.0) (concentration 7 mM in HBSS containing 10 mM HEPES, pH 7.4, 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, 4.75, 4.5 adjusted using NaOH), as a weak acid and strong acid, respectively. We did not use weaker acid like acetic acid (pKa: 4.76) than citric acid, because our pilot studies showed that acetic acid frequently induced nonspecific calcium responses in HEK293 cells in the absence of transfected receptor DNAs as reported previously²⁹. For the same reason, citric acid was limited in its usable pH (pH 5.0 or above). In all the experiments, we confirmed that acid solutions did not produce calcium responses before application of MCL by means of single cell Ca²⁺ imaging. For the experiment of intracellular acidification with extracellular neutral pH, we used modified HBSS; 90 mM bicarbonate solution (in mM) 1.26 CaCl₂, 0.493 MgCl₂, 0.407 MgSO₄ 5.33 KCl 0.441 KH₂PO₄ 90 NaHCO₃ 48 NaCl 0.338 Na₂HPO₄ 10 HEPES and 27.5 mM acetate solution (in mM) 1.26 CaCl₂, 0.493 MgCl₂, 0.407 MgSO₄ 5.33 KCl 0.441 KH₂PO₄ 4.17 NaHCO₃ 110 NaCl 0.338 Na₂HPO₄ 27.5 CH₃COONa 10 HEPES and pH were adjusted to 7.2 by titration with HCl²². For the calibration of intracellular pH using SNARF, we used modified HBSS (in mM): 1.26 CaCl₂, 0.493 MgCl₂, 0.407 MgSO₄ 140 KCl 0.441 KH₂PO₄ 4.17 NaHCO₃ 0.338 Na₂HPO₄ 10 HEPES: with 10 μM nigericin and pH was adjusted to 7.6, 7.4, 7.2, 7.0, 6.8, 6.6, 6.4, 6.2 with NaOH or HCl. Regents were purchased from Nacalai tesque (Isoproterenol), Ajinomoto (Aspartame) and Wako Pure Chemical Industries (others). MCL was purified from pulps of the miracle fruits (Richadella dulcifica) as reported previously²⁹.