The cystic fibrosis mutation G551D alters the non-Michaelis-Menten behavior of the cystic fibrosis transmembrane conductance regulator (CFTR) channel and abolishes the inhibitory Genistein binding site.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) channel activity explains most of the manifestations of the cystic fibrosis (CF) disease. To understand the consequences of CF mutations on CFTR channel activity, we compared the pharmacological properties of wild-type (wt) and G551D-CFTR. Dose-dependent relationships of wt-CFTR activated by genistein follows a non-Michaelis-Menten behavior consistent with the presence of two binding sites. With phosphorylated CFTR, a high affinity site for genistein is the activator (K(s) approximately 3 microm), whereas a second site of low affinity (K(i) approximately 75 microm) is the inhibitor. With non-phosphorylated CFTR, K(s) was increased (K(s) approximately 12 microm), but K(i) was not affected (K(i) approximately 70 microm). In G551D-CFTR cells, channel activity was recovered by co-application of forskolin and genistein in a dose-dependent manner. A further stimulation of G551D-CFTR channel activity was measured at concentrations from 30 microm to 1 mm. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K(m) approximately 11 microm). Our results suggest glycine 551 in NBD1 as an important location within the low affinity inhibitory site for genistein and offers new evidence for pharmacological alteration caused by an NBD1 mutation of CFTR. This study also reveals how a mutation of an ion channel converts a non-Michaelis-Menten behavior (two binding sites) into a classical Michaelis-Menten model (one binding site).