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Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The most common mutation, a deletion of the phenylalanine at position 508 ([DELTA]F508), disrupts processing of the protein. Nearly all human CFTR-[DELTA]F508 is retained in the endoplasmic reticulum and degraded, preventing maturation to the plasma membrane. In addition, the F508 deletion reduces the activity of single CFTR channels. Human CFTR-[DELTA]F508 has been extensively studied to better understand its defects. Here, we adopted a cross-species comparative approach, examining human, pig, and mouse CFTR-[DELTA]F508. As with human CFTR-[DELTA]F508, the [DELTA]F508 mutation reduced the single-channel activity of the pig and mouse channels. However, the mutant pig and mouse proteins were at least partially processed like their wild-type counterparts. Moreover, pig and mouse CFTR-[DELTA]F508 partially restored transepithelial [Cl.sup.-] transport to CF airway epithelia. Our data, combined with earlier work, suggest that there is a gradient in the severity of the CFTR-[DELTA]F508 processing defect, with human more severe than pig or mouse. These findings may explain some previously puzzling observations in CF mice, they have important implications for evaluation of potential therapeutics, and they suggest new strategies for discovering the mechanisms that disrupt processing of human CFTR-[DELTA]F508. airway epithelia | chloride transport | cystic fibrosis | mouse models

Research demonstrates that the regulation of channel activity of the cystic fibrosis transmemebrane conductance regulator (CFTR), an anion channel protein of the ABC transporter family, is regulated by ATPase and Adenylate kinase. However, at physiologic nucleotide concentrations, the latter enzyme controls gating.

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Am J Physiol Cell Physiol 281: C1657-C1666, 2001.--We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (E[C.sub.50] = 85 [micro]M) and activated CFTR in Calu-3 cells (E[C.sub.50] = 47 [micro]M). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity. pharmacology; disease-causing mutation; cystic fibrosis; nucleotide binding domains; cystic fibrosis transmembrane conductance regulator

Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-ΔF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and ΔF508 channels. Adding curcumin also increased Cl- transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl- channels. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P°,P5-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl- channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. [ABSTRACT FROM AUTHOR]

Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the CF transmembrane conductance regulator ( CFTR ) gene fulfills that goal. Integrating lentiviral vectors are well suited for this purpose, but widespread airway transduction in humans is limited by achievable titers and delivery barriers. Since airway epithelial cells are interconnected through gap junctions, small numbers of cells expressing supraphysiologic levels of CFTR could support sufficient channel function to rescue CF phenotypes. Here, we investigated promoter choice and CFTR codon optimization (co CFTR ) as strategies to regulate CFTR expression. We evaluated two promoters-phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α)-that have been safely used in clinical trials. We also compared the wild-type human CFTR sequence to three alternative co CFTR sequences generated by different algorithms. With the use of the CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK and identified a co CFTR sequence that conferred significantly increased functional CFTR expression. Optimized promoter and CFTR sequences advance lentiviral vectors toward CF gene therapy clinical trials., Competing Interests: P.B.M. is on the Scientific Advisory Board and receives support for sponsored research from Spirovant Sciences, Inc., (© 2021 The Author(s).)