Ion transport and pH homeostasis in coccolithophores
Most metabolic processes are pH dependent. If we want to understand the influence of ocean pH and carbonate chemistry on coccolithophores, it is necessary to gain a better understanding of their physiological properties and metabolic processes. Here Emiliania huxleyi and Coccolithus pelagicus were chosen to characterise some mechanisms involved in pH homeostasis and ion transport. Effects of changes in seawater carbon chemistry on intracellular pH (pHi) were measured by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)fluorescence. Out of equilibrium (OOE) solutions were used to differentiate between membrane permeation pathways for H+, CO2 and HCO3-. The ionophore nigericin was used to calibrate the dimension of changes in pHi measured by BCECF. pHi acutely followed the pH of seawater (pHe) in a linear fashion between pHe 6.5 and 9. No pHi change could be detected when seawater [CO2], [CO2]e, was increased at constant pHe and extracellular [HCO3-], [HCO3-]e. An increase in [HCO3-]e resulted in a slight intracellular acidification. In the presence of 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) pHi in E. huxleyi acidified and the effect was not reversible. In addition, DIDS reduced the effect of pHe on pHi slightly. The data for the first time show the occurrence of a direct proton permeation pathway in E. huxleyi plasma membrane, a direct acidifying impact of increased [HCO3-]e on pHi and no detectable influence of increased [CO2]e on pHi. pH homeostasis involves a DIDS sensitive mechanism. The data suggest the involvement of ion transport mechanisms which link ocean seawater pH and metabolic processes in E. huxleyi. To further characterise these mechanisms the impact of manipulated extracellular ion concentrations on pHi was investigated. The data on increased external [K+], [K+]e , and decreased external [Cl-], [Cl-]e, i. e. the effect of decreased gradients on pHi , showed more complex relationships. Both led to a first rapid but transient acidification of pHi and a second slower, also transient acidification upon return to control conditions. The two pH reactions showed different kinetics. The results indicate coupling of H+ transport to ion gradients. Different methods to isolate pure protoplasts and perform electrophysiological measurements on E. huxleyi were applied. E. huxleyi protoplasts showed a clean cell membrane by different methods; however the cells did not form gigaseals. In C. pelagicus protoplast isolation and sealing were achieved, however, only in limited numbers of cells. A revision of E. huxleyi membrane anatomy by confocal microscopy in collaboration with M. Gutowska and N. Fischer gave first evidence for a dual protoplast outer membrane, which might explain the difficulties in dye loading and patch sealing. In summary the collected data are a first step in characterising physiological properties of coccolithophores with respect to carbon transport pathways, and pH homeostasis on a cellular level.