REGULATION OF CFTR CL- CONDUCTANCE IN SECRETION BY CELLULAR-ENERGY LEVELS

Recent studies suggested dual regulation of the Cl- conductance (G(Cl)) affected in cystic fibrosis, one by protein kinase A-dependent phosphorylation and a second by low-affinity ATP binding. We proposed that ATP binding may couple the transport demands to the energy level of the cell. In the present study we examined this hypothesis further in a purely secretory function using the epithelial cell line T84. We used a depletion-permeabilization protocol on cells grown on permeable supports to deplete the cells of endogenous ATP and to provide access to the intracellular compartment for the impermeable nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and ATP. In contrast to non-depleted permeabilized cells, which responded to 0. 1 mM cAMP with an increase in transepithelial potential (DELTAV(t) = 29.8 +/- 3.0 mV, n = 4) and conductance (DELTA G(t) = 1.23 +/- 0.54 MS/cm2, n = 4), addition of cAMP to ATP-depleted cells resulted in insignificant changes in V(t) (DELTAV(t) = 0.7 +/- 0.2 mV, n = 26; P < 0.05) and G(t) (DELTAG(t) = 0.020 +/;- 0.003 mS/CM2, n = 26; P < 0.05). However, the cAMP response was restored by addition of 5 mM ATP (DELTAV(t) = 21.7 +/- 1.5 mV, n = 26; DELTAG(t) = 0.59 +/- 0.06 MS/CM2, n = 26). ATP dose-response experiments, taken together with the effect of cAMP with and without ATP, suggest that phosphorylation is necessary, but not sufficient, for activation. The data provide evidence for a second level of regulation of G(Cl), which requires high concentrations of ATP. Results using a nonhydrolyzable analogue of ATP suggest that this role may involve a nonhydrolytic interaction of ATP with the cystic fibrosis transmembrane conductance regulator.