CYTOSOLIC PH REGULATES G(CL) THROUGH CONTROL OF PHOSPHORYLATION STATES OF CFTR

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (G(Cl)). We monitored CFTR G(Cl) in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (V-C l) and transepithelial conductance (G(Cl)). We found that luminal pH ( 5.0-8.5) had little effect on the cAMP/ATP-activated CFTR G(Cl), showi ng that CFTR G(Cl) is maintained over a broad range of extracellular p H in which it functions physiologically. However, we found that phosph orylation activation of CFTR G(Cl) is sensitive to intracellular pH. T hat is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphos phate)], CFTR could be phosphorylated at physiological pH (6.8) but no t at low pH (similar to 5.5). On the other hand, basic pH prevented en dogenous phosphatase(s) from dephosphorylating CFTR. After phosphoryla tion of CFTR with cAMP and ATP, CFTR G(Cl) is normally deactivated wit hin 1 min after cAMP is removed, even in the presence of 5 mM ATP. Thi s deactivation was due to an increase in endogenous phosphatase activi ty relative to kinase activity, since it was reversed by the reapplica tion of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR G(Cl) in a dose-dependent manner, in dicating inhibition of dephosphorylation. We conclude that CFTR G(Cl) may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH pr obably has little direct effect on these mechanisms. This regulation o f CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.