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 (ΔR708 -759), R760 -783, and R784 - 835, each of which contains one or two PKA phosphorylation sites. Deletion of R708 -759 or R760 -783 had little effect on CFTR gating, whereas all mutations lacking R784 - 835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784 - 835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784 - 835 had minor impact on gating potentiation by 2'dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809 - 835 shortened the IBI of ΔR708 - 835-CFTR. Consistently, ΔR815- 835, but not ΔR784 - 814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784 - 835 is required for stabilizing the open state of CFTR; however, R815- 835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708 -783-CFTR. Conversely, the IBI of ΔR708 - 814-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784 - 814-mediated autoinhibition that prevents IBI shortening by R815- 835. This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.
CITATION STYLE
Chen, J. H. (2020). Protein kinase A phosphorylation potentiates cystic fibrosis transmembrane conductance regulator gating by relieving autoinhibition on the stimulatory C terminus of the regulatory domain. Journal of Biological Chemistry, 295(14), 4577–4590. https://doi.org/10.1074/jbc.RA119.008427
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