Direct Monitoring of Bicarbonate Transport by Emission Spectroscopy
The transmembrane transport of bicarbonate is a key step in many important biological processes, while problems with bicarbonate transport are at the origin of various diseases. Over the past 10 years, many anionophores that have been developed for the transport of chloride, have also been tested as bicarbonate transporters. However, methodology to directly monitor the kinetics of transport of bicarbonate is lacking, hence indirect methods have been used, which mainly rely on the monitoring of chloride concentrations.Here we present an assay that allows the kinetics of bicarbonate transport into liposomes to be monitored directly, using emission spectroscopy. The assay utilises an encapsulated europium(III) complex, which exhibits a large increase in emission upon binding of bicarbonate. The advantages of this assay over existing methodology are that concentrations of bicarbonate are monitored directly and with a high sensitivity. This allows studies at very low concentrations of anionophores, and for the mechanisms of bicarbonate transport to be unravelled. We have distinguished classical antiport with bicarbonate from mechanisms involving CO2 diffusion and the dissipation of a pH gradient. Furthermore, the use of a standard fluorescence spectrometer and liposomes with a diameter ~200 nm makes this assay readily and reliably applicable in many laboratories, where it can facilitate the development of bicarbonate transporters for applications in physiological studies or therapies.
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