In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer.
The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.
Mense
Martin
M
Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY 10021, USA.
Vergani
Paola
P
White
Dennis M
DM
Altberg
Gal
G
Nairn
Angus C
AC
Gadsby
David C
DC
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011387:Prokaryotic Cells / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014981:Xenopus; D014982:Xenopus laevis
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases