Abstract
Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. However, so far, the measurement of bicarbonate concentration changes in living cells has only been possible by measuring intracellular pH changes. In this article, we report the development of a genetically encoded pH-independent fluorescence-based single-use sensory cellular test system for monitoring intracellular bicarbonate concentration changes in living cells. We describe the usefulness of the developed biosensor in characterizing the bicarbonate transport activities of anionophores—small molecules capable of facilitating the membrane permeation of this anion. We also demonstrate the ability of the bicarbonate sensory cellular test system to measure intracellular bicarbonate concentration changes in response to activation and specific inhibition of wild-type human CFTR protein when co-expressed with the bicarbonate sensing and reporting units in living cells. A valuable benefit of the bicarbonate sensory cellular test system could be the screening of novel anionophore library compounds for bicarbonate transport activity with efficiencies close to the natural anion channel CFTR, which is not functional in the respiratory epithelia of cystic fibrosis patients.
Highlights
Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis
The bicarbonate sensory cellular test system is expressed in Chinese hamster ovary (CHO)-K1 cells
An increase in intracellular bicarbonate concentration activates sACtr resulting in an increase in intracellular cAMP concentration. cAMP binds to the regulatory domain of CEPAC resulting in a decrease in FRET intensity (Figure 2)
Summary
Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis.Regulation of bicarbonate transport across cell membranes is of critical importance.Bicarbonate is a labile molecule involved in several pH-dependent equilibria (Figure 1). Bicarbonate plays a central role in human physiology from cellular respiration to pH homeostasis. Regulation of bicarbonate transport across cell membranes is of critical importance. Bicarbonate is a labile molecule involved in several pH-dependent equilibria (Figure 1). At air–liquid interfaces, as in the lungs, gaseous CO2 is in equilibrium with dissolved CO2. The enzyme carbonic anhydrase (CA) catalyzes the reversible reaction of water and CO2 to form carbonic acid, which in turn is in equilibrium with bicarbonate. CA is a ubiquitous enzyme found in most organisms
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