Regulation of CFTR Cl- conductance in secretion by cellular energy levels
Bell, C.L.; Quinton, P.M.
American Journal of Physiology 264(4 Pt 1): C925-C931
1993
ISSN/ISBN: 0002-9513 PMID: 7682778 Document Number: 419902
Recent studies suggested dual regulation of the Cl- conductance (GCl) 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 (DVt = 29.8 ± 3.0 mV, n = 4) and conductance (dGt = 1.23 ± 0.54 mS/cm2, n = 4), addition of cAMP to ATP-depleted cells resulted in insignificant changes in Vt (DVt = 0.7 ± 0.2 mV, n = 26; P < 0.05) and Gt (DGt = 0.020 ± 0.003 mS/cm2, n = 26; P < 0.05). However, the cAMP response was restored by addition of 5 mM ATP (DVt = 21.7 ± 1.5 mV, n = 26; DGt = 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 GCl, 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. Reprinted by permission of the publisher.