Abstract
The epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP-dependent protein kinase (PKA). PKA stimulates CFTR channels through two mechanisms: non-catalytically, by binding to the channel, and catalytically, by phosphorylating its regulatory (R) domain. CFTR mutations that reduce channel activity cause cystic fibrosis (CF), but clinically used modulator drugs that boost channel function can alleviate disease symptoms. The two common CF mutations, ΔF508 and G551D, have been reported to impair CFTR channel activation by PKA, but the mechanisms remain unclear. Here, we aimed to understand how the mutations impact non-catalytic vs. catalytic channel activation by PKA and how these two processes are modulated by clinically used potentiator drugs. Using current recordings from excised inside-out membrane patches superfused with the purified catalytic subunit of PKA, we confirm slowed PKA-dependent activation for both mutants but demonstrate intact binding affinity for the kinase. Furthermore, we find that non-catalytic activation dominates overall channel activity for both mutants and can be strongly enhanced by stabilization of the NBD1-NBD2-TMD interface using the ATP analogue N(6)-(2-phenylethyl)-ATP. For both mutants, the clinically used potentiator drug combination elexacaftor + ivacaftor boosts catalytic channel activation by PKA more efficiently than non-catalytic activation. For ΔF508 CFTR, elexacaftor + ivacaftor evokes substantial PKA-independent channel activity and entirely suppresses non-catalytic activation by PKA. These findings help us to understand the activation defects caused by two common CF mutations and suggest room for further improvement of potentiator drugs currently used in CF therapy. KEY POINTS: Protein kinase A (PKA) activates the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) through two mechanisms: non-catalytically, by binding to the channel, and catalytically, by phosphorylating its regulatory (R) domain. CFTR mutations cause cystic fibrosis (CF); the two common mutations, ΔF508 and G551D, reportedly also impair channel activation by PKA. We show here, for both mutants, that PKA-dependent activation is slowed despite intact binding affinity for the kinase, and that non-catalytic activation dominates overall channel activity and might be further enhanced by stabilization of the NBD1-NBD2-TMD interface. For ΔF508 CFTR, but not for G551D CFTR, a combination of clinically used potentiator drugs evokes substantial PKA-independent channel activity but suppresses non-catalytic activation by PKA. These findings help us to understand the activation defects caused by two common CF mutations and suggest room for further improvement of potentiator drugs currently used in CF therapy.