Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by PKA phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing 1 to 3 protein segments of the R domain, including residues 708-759 (ΔR), R and R, each of which contains one or two PKA phosphorylation sites. Deletion of R or R had little effect on CFTR gating, whereas all mutations lacking R reduced CFTR activity by decreasing the mean burst duration (MBD) and increasing the interburst interval (IBI). The data suggest that R plays a major role in stimulating CFTR gating. For ATP-associated regulation, ∆R had minor impact on gating potentiation by 2’dATP, CaATP and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R shortened the IBI of ΔR-CFTR. Consistently, ΔR, but not ΔR, enhanced IBI, whereas both reduced MBD. These data suggest that entire R is required for stabilizing the open state of CFTR; however, R through interactions with the channel is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR-CFTR. Conversely, the IBI of ΔR-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R-mediated auto-inhibition that prevents IBI shortening by R This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.