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.