Abstract
The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.
Highlights
CFTR belongs to the family of Adenosine 50-triphosphoribose magnesium (ATP) Binding Cassette (ABC) proteins (Riordan et al, 1989), and forms an anion selective channel which is activated by phosphorylation of its cytosolic regulatory (R) domain by cyclic AMP-dependent protein kinase (PKA) (Berger et al, 1991; Tabcharani et al, 1991)
To generate an aqueous solution exactly saturated with Vx-770, a small aliquot of crystals was added to an aqueous saline and, after vigorous shaking for 24 hr, visible crystals were removed by repeated filtering and microcrystals sedimented by centrifugation (Materials and methods)
The discovery of the CFTR potentiator Vx-770 has caused a major change in the therapy of cystic fibrosis (CF) patients, allowing to address for the first time the root cause of the disease
Summary
CFTR belongs to the family of ATP Binding Cassette (ABC) proteins (Riordan et al, 1989), and forms an anion selective channel which is activated by phosphorylation of its cytosolic regulatory (R) domain by cyclic AMP-dependent protein kinase (PKA) (Berger et al, 1991; Tabcharani et al, 1991). The most common CF mutation, deletion of phenylalanine 508 (DF508), is present in ~90% of patients and impairs both channel surface expression (Cheng et al, 1990) and open probability (Miki et al, 2010). The potentiator Vx-770 (Van Goor et al, 2009), identified by Vertex Pharmaceuticals using high-throughput screening, has proven successful and was approved by the FDA for the treatment of patients carrying G551D and other gating mutations (Ramsey et al, 2011). This breakthrough has demonstrated the feasibility of efficient causative CF therapy using small-molecule potentiators, at least for this subset of patients (
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