A Small-Molecule Modulator Interacts Directly with Phe508- CFTR to Modify Its ATPase Activity and Conformational Stability

The deletion of Phe-508 ( Phe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by smallmolecule “correctors”; once at the cell surface, small-molecule “potentiators” enhance the channel activity of Phe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of Phe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted Phe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of Phe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to Phe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of Phe508-CFTR. Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a member of the ATP binding cassette (ABC) superfamily of membrane proteins (Riordan et al., 1989). Together, ABC family members mediate the transport of a diverse range of substrates, with some family members working as exporters and some as importers. These activities depend on regulated interactions between their cytosolic nucleotide binding domains (NBDs) and membrane-spanning domains (MSDs). The NBDs of CFTR mediate ATP binding and ATPase activity like the other members of this family (Li et al., 1996; Ramjeesingh et al., 2008). However, CFTR is unique in that its membrane-spanning domains form a chloride-selective pore to mediate chloride conduction (Bear et al., 1992; Linsdell, 2006). ATP binding at the interface between NBD1 and NBD2 in CFTR is thought to promote opening of the channel gate, whereas ATPase activity promotes dissociation of the NBD heterodimer, leading to the closing of the channel (Lewis et al., 2004; Vergani et al., 2005a; Aleksandrov et al., 2007). Opening of the CFTR channel gate also requires phosphorylation of multiple serine residues within the unique “R domain” region by PKA, a modification that alters “R domain” interactions with other domains, notably NBD1 (Winter and Welsh, 1997; Seibert et al., 1999; Csanády et al., This work was funded by the Canadian Cystic Fibrosis Foundation, the Canadian Institute of Health Research (BREATHE PROGRAMME 1); by the Cystic Fibrosis Foundation Therapeutics Foundation [Grant BEAR06DDS0] (to C.B.); by a Natural Sciences and Engineering Research Council Postgraduate Doctoral Award (to L.W.); and by a Doctoral Award provided by the Canadian Institutes of Health Research Strategic Training Programme in the Structure of Membrane Proteins and Disease (to P.K.C.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.055608. ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP binding cassette; NBD, nucleotide binding domain; MSD, membrane-spanning domain; PKA, protein kinase A; VRT-532, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol; BHK, baby hamster kidney; HA, hemagglutinin; MOPS, 3-(N-morpholino)propanesulfonic acid; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; FL, full-length; CFTRinh-172, CFTR inhibitor-172. 0026-895X/09/7506-1430–1438$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 6 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 55608/3480388 Mol Pharmacol 75:1430–1438, 2009 Printed in U.S.A. 1430 at A PE T Jornals on D ecem er 9, 2017 m oharm .aspeurnals.org D ow nladed from 2005; Baker et al., 2007). Overall, interactions between multiple domains are necessary to communicate signals between cytosolic domains and the chloride channel gate in the mem-