The cystic Γbrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide binding domains (NBDs). It has been controversial whether CFTR conducts, or is otherwise permeable to ATP, or whether CFTR might be closely associated with a separate ATP conductance. The relevance of this issue derives from its implications regarding the ion channel properties of CFTR, the potential for released ATP to function as a local signal to activate other transport pathways, and the possibility that a CFTR-associated adenine nucleotide permeability may modulate glycoprotein processing.
To characterize ATP channels which might be associated with the expression of CFTR, we analyzed Cl -and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl -in the bath, ATP channels were associated with CFTR Cl -channels in two thirds of patches that included CFTR. CFTR Cl -channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl -channels. Gating kinetics of the ATP channels as well as the CFTR Cl - channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylationand nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl -and ATP are distinct, and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.
These data may rationalize the discrepant results among different laboratories regarding the ability to detect a CFTR-associated ATP permeability, because they suggest a cell-type dependence, and furthermore, a