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

Abstract

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. Dosedependent 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 Ϸ 3 M), whereas a second site of low affinity (K i Ϸ 75 M) is the inhibitor. With non-phosphorylated CFTR, K s was increased (K s Ϸ 12 M), but K i was not affected (K i Ϸ 70 M). 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 M to 1 mM. The dose response is described by a classical Michaelis-Menten kinetics with only a single apparent site (K m Ϸ 11 M). 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).

C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation

“…All experiments were performed and analyzed as described previously (17). Iodide efflux curves were constructed by plotting rate (k, min ).…”

A Rare Transporter Associated with Antigen Processing Polymorphism Overpresented in HLAlow Colon Cancer Reveals the Functional Significance of the Signature Domain in Antigen Processing

“…(11,13,14,30). As a first step to determine whether the polymorphism of signature motif region of TAP-1 contributes to variations in TAP-1function, we compared the HLA low and HLA high colon cancer samples for the DNA sequence in exon 10 which encodes this region.…”

Genetic, cell biological, and clinical interrogation of theCFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention

“…VX-770 (or Ivacaftor) has been approved in North America and Europe as a drug for patients bearing the relatively rare mutation: p.Gly551Asp. 7,8 The p.Gly551Asp mutation causes defective channel gating, [9][10][11] and VX-770 is thought to be effective in partially restoring lung function in patients with p.Gly551Asp because it enhances the channel activity of this mutant. [12][13][14][15] The most common mutation in Europe and North America, p.Phe508del, has been studied extensively and has been found to impair CFTR protein folding during synthesis.…”