Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

Abstract

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

“…Phosphorylation reduces the bias toward helical conformations and decreases many structural contacts within the R region, except in the vicinity of Ser768. The decreases in helicity and R 2 relaxation rates on the N-terminal side of Ser737 are consistent with the marked gel shift previously observed upon phosphorylation at this site, which results from a conformational change 34 and may also reflect changes in nonlocal structure. 36 .…”

Mechanistic Insight into Control of CFTR by AMPK

“…S2). The gel shifts observed with PKA (but not AMPK) in the presence of these mutants are unexplained but are consistent with a selective change in R domain structure after PKA phosphorylation (24,25). Correspondingly, Fig.…”

“…Interestingly two of these sites, namely Ser 737 and Ser 768 , have been identified as "inhibitory" R domain sites, i.e. when mutated to alanines they augment the open probability of CFTR relative to wild type (23,24). We hypothesized that these sites might be phosphorylated by AMPK (rather than "inhibited" by PKA) given some sequence homology with the expected consensus sequence for AMPK in local amino acids and therefore examined in vitro phosphorylation of isolated single or double mutants or wild type R domain protein.…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…Fifteen sites must be eliminated to create CFTR channels that are completely PKA-insensitive (Seibert et al 1999;Hegedus et al 2009). Two PKA sites (S737 and S768) are argued to be inhibitory on the basis of the observation that their disruption increases channel activity (Wilkinson et al 1997;Csanády et al 2005). These sites also appear to be phosphorylated by AMP kinase which inhibits CFTR activity possibly under hypoxic conditions (King et al 2009).…”