More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.

The genetic disease cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel. Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Both mutants severely disrupt CFTR channel gating by decreasing mean burst duration (MBD) and prolonging greatly the interburst interval (IBI). To identify small molecules that rescue the gating defects of G551D-and G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551D-and G1349D-CFTR of phloxine B, pyrophosphate (PP i ), and 2 -deoxy ATP (2 -dATP), three agents that strongly enhance CFTR channel gating. Phloxine B (5 M) potentiated robustly G551D-CFTR Cl ؊ channels by altering both MBD and IBI. In contrast, phloxine B (5 M) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. PP i (5 mM) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl ؊ channel. However, by altering both MBD and IBI, albeit with different efficacies, 2 -dATP (1 mM) potentiated both G551D-and G1349D-CFTR Cl ؊ channels. Using the ATPdriven nucleotide-binding domain dimerization model of CFTR channel gating, we suggest that phloxine B, PP i and 2 -dATP alter channel gating by distinct mechanisms. We conclude that G551D-and G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific.The cystic fibrosis transmembrane conductance regulator (CFTR 2 (1)) is a unique member of the ATP-binding cassette transporter superfamily that plays a critical role in fluid and electrolyte transport across epithelia (2). CFTR is composed of two membrane-spanning domain (MSD) nucleotide-binding domain (NBD) motifs linked by a unique regulatory (R) domain. The membrane-spanning domains assemble to form a transmembrane pore with deep intracellular and shallow extracellular vestibules that funnel anions toward a selectivity filter, which determines the permeation properties of CFTR (3). Anion flow through the CFTR pore is controlled by cycles of ATP binding and hydrolysis at two ATP-binding sites located at the interface of the two NBDs (4). Stable ATP binding occurs at one ATPbinding site (site 1; formed by the Walker A and B motifs of NBD1 and the LSGGQ motif of NBD2), whereas rapid ATP turnover occurs at the other ATP-binding site (site 2; formed by the Walker A and B motifs of NBD2 and the LSGGQ motif of NBD1) (5, 6). The R domain contains multiple consensus phosphorylation sites on the surface of an unstructured domain (7). Phosphorylation of the R domain stimulates CFTR function by enhancing ATP-dependent channel gating at the NBDs (3). Thus, CFTR is an anion channel with exquisite regulation.The importance of CFTR for transepithelial ion transport is dramatically highlighted by its malfunction in human disease. The genetic disease CF is caused by muta...

The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wtand V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention͞retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control. endoplasmic reticulum quality control ͉ folding ͉ membrane traffic ͉ arginine-framed motifs ͉ trafficking signals

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wideranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside‐out membrane patches from cells expressing wild‐type human CFTR. Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single‐channel current amplitude (i), but a large reduction in open probability (Po). Single‐channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. Genistein inhibition of CFTR Cl− currents was weakly voltage dependent and unaffected by changes in the extracellular Cl− concentration. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide‐binding domains (NBDs) of CFTR to greatly stimulate channel activity. ATP (5 mM) prevented the genistein‐induced decrease in Po, but was without effect on the genistein‐induced decrease in i. The genistein‐induced decrease in i was voltage dependent, whereas the genistein‐induced decrease in Po was voltage independent. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl− permeation.