Fig. 1.

Cytosolic acidification of HvPIP2;1-injected X. laevis oocytes induced by perfusion of carbon dioxide-enriched buffer. (A) Typical raw recordings of water-injected and HvPIP2;1 cRNA-injected oocytes by perfusion with modified Barth’s solution, of which the NaCl and NaHCO₃ concentrations and pH were modified. The raw recordings represent the difference of the reading of two electrodes (V_pH – V_ref). V_pH and V_ref indicate the voltage reading of the hydrogen ion-selective microelectrode and the membrane potential microelectrode, respectively. The pH of the buffer was adjusted to 7.31, so that the ratio of CO₂/H₂CO₃ to was 0.1. The concentration of CO₂/H₂CO₃ was changed from 0.1 mM to 6.5 mM by perfusion. The perfusion was initiated where indicated by arrowheads. The buffer around the oocyte was replaced 10 s after the start of the perfusion in a typical measurement. This duration was estimation by pH change without an oocyte present. (B) The cytosolic pH change of water-injected (Water, green line) and HvPIP2;1 cRNA-injected (HvPIP2;1, magenta line) oocytes by perfusion with modified Barth’s solution, of which the NaCl and NaHCO₃ concentrations and pH were modified. The cytosolic pH was measured by hydrogen ion-selective microelectrodes. In the bath solution with 0.01 mM CO₂/H₂CO₃, NaHCO₃ was substituted with NaCl and the concentration of CO₂/H₂CO₃ was determined by equilibration with the ambient air. The bath solutions which included 0.22, 0.65, 2.2 and 6.5 mM CO₂/H₂CO₃ (CO₂-enriched buffer) were prepared by replacing NaCl with NaHCO₃ to give the appropriate CO₂/H₂CO₃ concentrations in the bath solution. The CO₂-enriched buffers were aliquoted and sealed with caps immediately after the preparation to prevent the diffusional loss of CO₂ gas into the air. The oocytes were equilibrated to the bath solution containing 0.01 mM CO₂/H₂CO₃ and impaled with the microelectrodes as described in the Materials and Methods. Subsequently, the bath solution was perfused with a peristaltic pump at a rate of 400 µl min⁻¹. Hum noise (60 Hz) was cancelled in silico. Note that the y-axis was converted from electric potential to pH according to calibration lines. A typical calibration line is shown in Supplementary Fig. S6. (C) Rate of cytosolic acidification of water-injected (Water) and HvPIP2;1 cRNA-injected (HvPIP2;1) oocytes as shown by the reciprocal of the time constant (1/τ). The CO₂/H₂CO₃ concentration in the bath solution was replaced by perfusion (400 µl min⁻¹) from 0.01 mM to 6.5 mM (0.01→6.5) or 2.2 mM (0.01→2.2). τ was determined by exponential curve fitting. Water (0.01→6.5), n = 11. HvPIP2;1 (0.01→6.5, n = 10. Water (0.01→2.2), n = 7. HvPIP2;1 (0.01→2.2), n = 6. Asterisks indicate a significant difference of the mean of HvPIP2;1 from that of the water control at α = 0.05.