Protein kinase A phosphorylation potentiates cystic fibrosis transmembrane conductance regulator gating by relieving autoinhibition on the stimulatory C terminus of the regulatory domain

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (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 one to three protein segments of the R domain including residues 708–759 (ΔR 708–759 ), R 760–783 , and R 784–835 , each of which contains one or two PKA phosphorylation sites. Deletion of R 708–759 or R 760–783 had little effect on CFTR gating, whereas all mutations lacking R 784–835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R 784–835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR 784–835 had minor impact on gating potentiation by 2′dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R 809–835 shortened the IBI of ΔR 708–835 -CFTR. Consistently, ΔR 815–835 , but not ΔR 784–814 , enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R 784–835 is required for stabilizing the open state of CFTR; however, R 815–835 , through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR 708–783 -CFTR. Conversely, the IBI of ΔR 708–814 –CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R 784–814 –mediated autoinhibition that prevents IBI shortening by R 815–835 . This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.