Cl. Bell et Pm. Quinton, REGULATION OF CFTR CL- CONDUCTANCE IN SECRETION BY CELLULAR-ENERGY LEVELS, The American journal of physiology, 264(4), 1993, pp. 925-931
Recent studies suggested dual regulation of the Cl- conductance (G(Cl)
) affected in cystic fibrosis, one by protein kinase A-dependent phosp
horylation and a second by low-affinity ATP binding. We proposed that
ATP binding may couple the transport demands to the energy level of th
e 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 supp
orts to deplete the cells of endogenous ATP and to provide access to t
he intracellular compartment for the impermeable nucleotides adenosine
3',5'-cyclic monophosphate (cAMP) and ATP. In contrast to non-deplete
d permeabilized cells, which responded to 0. 1 mM cAMP with an increas
e in transepithelial potential (DELTAV(t) = 29.8 +/- 3.0 mV, n = 4) an
d 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 w
as 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 exper
iments, taken together with the effect of cAMP with and without ATP, s
uggest that phosphorylation is necessary, but not sufficient, for acti
vation. The data provide evidence for a second level of regulation of
G(Cl), which requires high concentrations of ATP. Results using a nonh
ydrolyzable analogue of ATP suggest that this role may involve a nonhy
drolytic interaction of ATP with the cystic fibrosis transmembrane con
ductance regulator.