Simple binding of protein kinase A prior to phosphorylation allows CFTR anion channels to be opened by nucleotides

Abstract

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt–water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt–water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches––an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines––that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA––and possibly also to other members of a large network of proteins known to interact with the channel.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP‐binding site

“…1 in Mihályi et al 2016;Fig. 4 in Mihályi et al 2020), we noticed phosphorylation-independent rundown. After CFTR channels are deprived of ATP for a prolonged period of time, upon readdition of ATP, CFTR currents rise in two phases -a fast one completed within seconds followed by a slower rising phase that can last for tens to hundreds of seconds (e.g.…”

Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation

“…closed NBD dimer, and thereby to the initial conformational changes driven by the PKA-dependent phosphorylation of the R domain (Liu et al, 2017;Mihalyi et al, 2020).…”

“…Of note, Liu and colleagues with the resolution of the human CFTR structure reveal a previously unresolved helix belonging to the R region docked in an inward-facing conformation between the two halves of CFTR, where it acts as a steric block precluding channel opening ( Liu et al, 2017 ). On the other hand, activation of the CFTR channel is strictly coupled to the formation of a closed NBD dimer, and thereby to the initial conformational changes driven by the PKA-dependent phosphorylation of the R domain ( Liu et al, 2017 ; Mihalyi et al, 2020 ).…”

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

“…Although the CFTR-mediated anion secretion is often dependent on protein kinase A (PKA), phosphoinositide 3-kinase (PI3K) or an increase in intracellular calcium ([Ca] i ) [ [9] , [10] , [11] ], it is unclear whether PTH directly alters PKA/PI3K phosphorylation or [Ca] i all of which are crucial for CFTR activity. Meanwhile, NHE1—known to be abundantly expressed in the basolateral membrane of enterocytes—is postulated to help maintaining normal pH i during anion secretion [ [12] , [13] , [14] ].…”