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Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain
Journal article   Open access   Peer reviewed

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain

Devra P Rich, Herbert A Berger, Seng H Cheng, Sue M Travis, Mumtaz Saxena, Alan E Smith and Michael J Welsh
The Journal of biological chemistry, Vol.268(27), pp.20259-20267
1993
DOI: 10.1016/S0021-9258(20)80723-6
PMID: 7690753
url
https://doi.org/10.1016/S0021-9258(20)80723-6View
Published (Version of record) Open Access

Abstract

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.
 Fundamental and applied biological sciences. Psychology Proteins Binding and carrier proteins Biological and medical sciences Analytical, structural and metabolic biochemistry

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