Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH_i). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H⁺, CO₂ and HCO₃⁻. Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH_i unit. pH_i acutely followed the pH of seawater (pH_e) in a linear fashion between pH_e values of 6.5 and 9 with a slope of 0.44 per pH_e unit. pH_i was nearly insensitive to changes in seawater CO₂ at constant pH_e and HCO₃⁻. An increase in extracellular HCO₃⁻ resulted in a slight intracellular acidification. In the presence of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH_e on pH_i. The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH_i homeostasis involves a DIDS-sensitive mechanism.