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

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 (pH_i) would alter the binding affinity. Here, we analyzed the effect of pH_i on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH_i does modify CFTR maximum current (I_m) and half-activation concentration (K_d): I_m = 127.7, 185.5, and 231.8 μA/cm² and K_d = 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 (K_a) increased at alkaline pHi, near cysteine pK (K_a = 1.83, 1.81 and 4.99 μM at pH_i 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 pH_i. Effects of pH_i on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH_i on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.