Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl ؊ channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 M) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is ϳ60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.
Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.
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