Figure 5. H⁺ conductance-mediated regulation of pH_i.

(A) Representative simultaneous whole cell patch clamp and pH_i imaging in C. pelagicus cells (300 µM BCECF free acid loaded via the patch pipette). Top panel displays false colour BCECF fluorescence ratio images of C. pelagicus during the voltage step protocol (+20, −50, and +70 mV). The inset (top right) indicates localization of BCECF (green), chlorophyll autofluorescence (red), and reflectance of an internal developing coccolith (white). Scale bar, 10 µm. Membrane depolarization to +20 mV or +70 mV from a holding potential of −50 mV caused an increase in pH_i. The pipette solution contained (in mM) K-Glutamate 200, MgCl₂ 5, EGTA 5 and Pipes 1.0 (P1b, Table S2), and the external solution was ASW containing (in mM) NaCl 450, KCl 8, MgCl₂ 30, MgSO₄ 16, CaCl₂ 10, NaHCO₃ 2, and HEPES 20 (E1, Table S2). (B) The effect of pH_o on pH_i in BCECF-loaded C. pelagicus cells. Changing pH_o from 8.0 to 6.5 induced a substantial and reproducible reduction in pH_i. Traces from 4 individual cells are superimposed. (C) The effect of Zn²⁺ on pH_i in calcifying C. pelagicus cells. Cells loaded with BCECF-AM were perfused with either f/2 FSW media pH 8.0 (n = 62) or artificial seawater (ASW) pH 8.0, containing 0 mM or 10 mM Ca²⁺ (n = 28 and 7, respectively). 30 µM free Zn²⁺ was perfused for 2.5 min (grey box); averaged traces are shown.