Abstract
Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin-Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a pK a value of 6.5±0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H+ efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo.
Original language | English |
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Pages (from-to) | 233-242 |
Number of pages | 10 |
Journal | Pflugers Archiv European Journal of Physiology |
Volume | 457 |
Issue number | 1 |
DOIs | |
State | Published - 2008 |
Externally published | Yes |
Bibliographical note
Funding Information:This work was supported by Fondecyt grant 1051081. The Centro de Estudios Científicos is funded by an Institute grant from the Millennium Science Initiative and by Centers of Excellence Base Financing Program, Conicyt and Gobierno Regional de Los Ríos. CECS is also supported by the following corporate benefactors: Antofagasta Minerals, Arauco, Empresas CMPC, Indura, Naviera Ultragas, and Telefónica del Sur. We are grateful to Dr. Jennifer Carbrey, Duke University Medical Center for providing the rat AQP4 plasmid, Dr. David Sheppard, University of Bristol for providing MDCK-1 cells, and Dr. Wolf Frommer, Carnegie Institution for kind donation of plasmids. The help of Gaspar Peña in the construction of the perfusion chamber used in confocal studies is gratefully acknowledged. We are grateful to Dr. Rafael Burgos, Universidad Austral de Chile for generously providing access to equipment.
ASJC Scopus subject areas
- Physiology
- Clinical Biochemistry
- Physiology (medical)