The ATPase Activity of CFTR Measured in Living Cells

Abstract

The cystic fibrosis transmembrane conductance regulator CFTR (ABCC7) is a homolog of the adenosine triphosphate (ATP) binding cassette (ABC) transporter P-glycoprotein (ABCB1) and functions as a chloride channel. Opening of the chloride channel requires ATP binding at the nucleotide binding domains and is enhanced by phosphorylation of the regulatory domain. Chloride flux is generally monitored by means of the patch clamp technique. Here we show that ATPase activity can be easily measured in living cells by monitoring the extracellular acidification rate (ECAR) using silicon chip technology as shown previously for P-glycoprotein. The ECAR of CFTR transfected Chinese hamster ovary (CHO) and the corresponding wild type cells, respectively, were measured under different phosphorylation conditions, using CPT-cAMP and forskloin. Whereas wild type cells showed only negligibly small effects, the ECAR of CFTR transfected cells increased at low concentrations, reached a maximum (up to 180% of the basal value), and decreased again at high concentrations, yielding bell-shaped activity curves. The lactate concentration was assessed in parallel, revealing that the extracellular acidification was due entirely to the extrusion of lactate which is a byproduct of anaerobic ATP synthesis. As ATP synthesis directly correlates with ATP hydrolysis the present approach allows a quantitative estimate of the rate ATP hydrolysis by CFTR under the different phosphorylation conditions. Acknowledgment: We are grateful to Prof. J. R. Riordan (University of North Carolina, USA) for providing the CHO cell overexpressing CFTR.