Investigating the Effect of PKA Phosphorylation on Intramolecular Interactions in Purified Full Length Wildtype CFTR

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an unique anion channel of the ATP-Binding Cassette (ABC) superfamily. CFTR consists of two nucleotide binding domains (NBDs), two membrane spanning domains (MSDs) and a regulatory (R) domain. There are also alpha-helical extensions and intracellular loops (ICLs) which couple the NBDs and MSDs via “coupling helices”. The regulation CFTR channel activity involves protein kinase A (PKA) phosphorylation at the R domain. However, not much is known about the effect of phosphorylation on the intramolecular interactions of full length CFTR. We propose that phosphorylation modifies the interactions of full length CFTR, especially at the ICL4:NBD1 interface. Previous studies of a similar ABC transporter, BtuCD, have shown that intrinsic tryptophan fluorescence can detect urea sensitive changes in the coupling between the MSDs and NBDs of full length BtuCD. Thus, we decided monitor intrinsic tryptophan fluorescence to study the effect of phosphorylation on the purified full length Wt CFTR. Interestingly, we found that the urea sensitive changes in the intrinsic tryptophan fluorescence of full length CFTR were modified upon PKA phosphorylation. We were interested to determine whether this result was due to phosphorylation modifying the ICL4:NBD1 interface. In order to specifically study that interface, cysteine crosslinking studies were employed using a short cell permeable cysteine crosslinker on a cys-less CFTR mutant with two cysteines, V510C (NBD1) and A106C7C (ICL4), transfected in HEK293 cells. Phosphorylation induced by cAMP agonists in cells resulted in a significantly increased the crosslinking of cysteines at the ICL4:NBD1 interface compared to the inhibition of phosphorylation with adenyl cyclase inhibitors. This suggests that phosphorylation modifies the ICL4 and NBD1 interface. These studies further our understanding of the molecular mechanisms underlying phosphorylation dependent gating of CFTR.