Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulated by three cytosolic domains, the regulatory domain (R domain) and two nucleotide binding domains. To learn more about how the cytosolic domains regulate channel activity, we used chemical modification to probe their structure. When we applied the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) and other N-substituted maleimides to the cytosolic domains, we found that they rapidly and irreversibly stimulated channel activity. CFTR contains 14 intracellular cysteine residues that might be targets for NEM modification. We identified one, Cys832, that was essential for the response. Cys832 is located in the R domain. Single channel studies showed that NEM stimulated CFTR by increasing the duration of bursts of activity and by shortening the closed interval between bursts. At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Additional studies showed that NEM modification increased the potency of ATP-mediated stimulation. Previous work has shown that modification of the R domain by phosphorylation, which introduces negative charge, or replacement of multiple serines by negatively charged aspartates stimulates the channel. Our current data show that covalent modification of the R domain with a neutral, hydrophobic adduct at a site that is not phosphorylated can also stimulate CFTR. This finding suggests that an alteration in the conformation of the R domain may be a key feature that regulates channel activity.