Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH

Raffaella Melani, Valeria Tomati, Luis J V Galietta, Olga Zegarra-Moran

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Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pHi) would alter the binding affinity. Here, we analyzed the effect of pHi on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pHi does modify CFTR maximum current (Im) and half-activation concentration (Kd): Im = 127.7, 185.5, and 231.8 μA/cm2 and Kd = 32.7, 56.6 and 71.9 μM at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (Ka) increased at alkaline pHi, near cysteine pK (Ka = 1.83, 1.81 and 4.99 μM at pHi 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pHi. Effects of pHi on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pHi on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.

Original languageEnglish
Pages (from-to)41591-41596
Number of pages6
JournalJournal of Biological Chemistry
Issue number53
Publication statusPublished - Dec 31 2010

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology


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