The human CFTR protein expressed in CHO cells activates an aquaporin 3 in a cAMP dependent pathway: study by Digital Holographic Microscopy

Abstract

The transmembrane water movements during cellular processes and their relation to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to CFTR (Cystic Fibrosis Transmembrane conductance Regulator) activity through a cAMP-stimulated aqueous pore or dependent on aquaporin. Here, we used the Digital Holographic Microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel CFTR measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh-172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR, F508del-CFTR, mimicking the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators genistein and MPB91 failed to trigger water movements. Finally, using a specific small-interfering RNA (siRNA) against the endogenous aquaporin AQP3, the water transport accompanying the CFTR activity decreased. We conclude that water fluxes accompanying the CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in CFTR protein.