BACKGROUND:Perturbation of endothelial function in people with cystic fibrosis (CF) has been reported, which may be associated with endothelial cell expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Previous reports indicate that CFTR activity upregulates endothelial barrier function, endothelial nitric oxide synthase (eNOS) expression and NO release, while limiting interleukin-8 (IL-8) release, in human umbilical vein endothelial cells (HUVECs) in cell culture. In view of reported microvascular dysfunction in people with CF we investigated the role of CFTR expression and activity in the regulation of oxidative stress, cell signaling and inflammation in human lung microvascular endothelial cells (HLMVECs) in cell culture.METHODS:HLMVECs were cultured in the absence and presence of the CFTR inhibitor GlyH-101 and CFTR siRNA. CFTR expression was analyzed using qRT-PCR, immunocytochemistry (IHC) and western blot, and function by membrane potential assay. IL-8 expression was analyzed using qRT-PCR and ELISA. Nrf2 expression, and NF-κB and AP-1 activation were determined using IHC and western blot. The role of the epidermal growth factor receptor (EGFR) in CFTR signaling was investigated using the EGFR tyrosine kinase inhibitor AG1478. Oxidative stress was measured as intracellular ROS and hydrogen peroxide (H2O2) concentration. VEGF and SOD-2 were measured in culture supernatants by ELISA.RESULTS:HLMVECs express low levels of CFTR that increase following inhibition of CFTR activity. Inhibition of CFTR, significantly increased intracellular ROS and H2O2 levels over 30 min and significantly decreased Nrf2 expression by 70% while increasing SOD-2 expression over 24 h. CFTR siRNA significantly increased constitutive expression of IL-8 by HLMVECs. CFTR inhibition activated the AP-1 pathway and increased IL-8 expression, without effect on NF-κB activity. Conversely, TNF-α activated the NF-κB pathway and increased IL-8 expression. The effects of TNF-α and GlyH-101 on IL-8 expression were additive and inhibited by AG1478. Inhibition of both CFTR and EGFR in HLMVECs significantly increased VEGF expression. The antioxidant N-acetyl cysteine significantly reduced ROS production and the increase in IL-8 and VEGF expression following CFTR inhibition.CONCLUSION:Functional endothelial CFTR limits oxidative stress and contributes to the normal anti-inflammatory state of HLMVECs. Therapeutic strategies to restore endothelial CFTR function in CF are warranted.

Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life‐shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. AbstractCross‐species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single‐channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del‐CFTR, using excised inside‐out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA‐dependent phosphorylation, inhibited by the open‐channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single‐channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature‐sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP‐dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.

The anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) transporter. CFTR plays a pivotal role in transepithelial ion transport as its dysfunction in the genetic disease cystic fibrosis (CF) dramatically demonstrates. Phylogenetic analysis suggests that CFTR first appeared in aquatic vertebrates fulfilling important roles in osmosensing and organ development. Here, we review selectively, knowledge of CFTR structure, function and pharmacology, gleaned from cross-species comparative studies of recombinant CFTR proteins, including CFTR chimeras. The data argue that subtle changes in CFTR structure can affect strongly channel function and the action of CF mutations.

Background and PurposeLoop diuretics are widely used to inhibit the Na+, K+, 2Cl(-) co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade.Experimental ApproachUsing the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR.Key ResultsWhen added to the intracellular solution, loop diuretics inhibited CFTR Cl- currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl- channels with very fast' speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with intermediate' speed and drug-induced blocking events were distinct from channel closures.Conclusions and ImplicationsStructure-activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.

P2 receptors are potent modulators of key solute and water transport mechanisms in the renal collecting duct (CD) [1]. Controversy exists over the P2 receptor subtype(s) responsible; most studies focus on metabotropic P2Y receptors and arguably ionotropic P2X receptors are largely overlooked.Using an M1 cell line derived from mouse CD and the excised outside‐out configuration of the patch‐clamp technique we have sought functional evidence for extracellular ATP‐gated P2X receptor ion channels. External bath solution contained 145 mM NaCl and the pipette solution contained 145 mM K+ (70 mM Cl−).In outside‐out patches, application of external ATP (100 μM) transiently activated flickery single channel currents (tact ~3s, tdes ~20s). At −100 mV, the mean inward single channel current amplitude (i), open probability (Po) and chord conductance of channels were 0.71±0.12 pA, 0.66±0.12, and 7.7±0.8 pS (n=5), respectively.We demonstrate that ATP directly activates receptors with singlechannel properties similar to those of P2X receptors. Consequently P2X receptors may regulate CD solute and water transport.Research supported by the Wellcome Trust, BBSRC and DiscoveryBiomed.

This is the pre-peer reviewed version of the following article: Ju, M., Scott-ward, T. S., Liu, J., Khuituan, P., Li, H., Cai, Z., ... Sheppard, D. N. (2014). Loop diuretics are open-channel blockers of the cystic fibrosis transmembrane conductance regulator with distinct kinetics. British Journal of Pharmacology, 171(1), 265-278, which has been published in final form at 10.1111/bph.12458. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."

Urinary tract infections (UTIs) cause complications for renal transplant recipients (RTRs) and may result in graft loss. Routine urinalysis to detect UTIs is insensitive/inaccurate in RTRs. UTI detection methods with greater accuracy are needed. A ≥50 nmol/l urinary ATP concentration indicates UTI and ATP measurement may be a superior diagnostic test (1). We investigated various urinalysis tests in 53 RTRs. ATP concentrations and intracellular bacteria (IB) in shed urothelial cells were compared to bacterial culture results. 22% of RTRs tested UTI positive by bacterial culture. IB were identified in 44% or RTRs, but only 2% IB positive were also UTI positive by bacterial culture. There was a significant association of the conventional bacterial culture with both urinary ATP concentration and evidence of IB (Fisher's exact test, p<0.05). 6% of RTRs had ATP concentrations ≥50 nmol/l. Those positive were also bacteria positive. 77% patients with ATP concentrations <50 nmol/l were negative by bacteria culture. Diagnostic odds ratios (DORs) indicated that ATP was a much stronger UTI indicator (DOR = 30.580) compared to IB (0.079). However combined urinary ATP concentration and evidence of IB was an even stronger diagnostic indicator of UTI (DOR = 105). Low urinary ATP concentration combined with absence of IB in shed urothelial may provide as superior diagnostic test for absence of UTI in RTRs.Funding: St Peter's Trust (UK).

In the renal collecting duct (CD), an ionotropic P2X4‐like receptor may act as a luminal Na sensor, regulating ENaC‐mediated Na reabsorption [1]. Several P2X receptor subunits are expressed in the CD and evidence is emerging that P2X4 and P2X7 subunits might interact [2]. P2X7 is also linked to many renal pathologies.Here we determine whether P2X4 and P2X7 interact at the expression level in the CD. Using target‐specific primers, we quantified P2X4, P2X7 (and β‐actin) mRNA levels in mouse kidney and microdissected CD of wild‐type (WT), P2X4−/− and P2X7−/− mice by real‐time RT‐PCR.Expression of P2X4 and, to a lesser extent P2X7 mRNA was readily detected in WT kidney (n=3), whereas P2X4 and P2X7 mRNA levels were <100‐fold lower in P2X4−/− and P2X7−/− mice respectively (n=3). Expression of P2X4 mRNA was reduced in P2X7−/− kidney (P=0.03; n=3), and a substantial decrease in P2X7 mRNA was observed in the CD of P2X4−/− mice (P<0.05; n=3).Our studies indicate that expression levels of P2X4 and P2X7 in the collecting duct are interdependent and suggest a P2X4/7 heteromeric assembly as the luminal Na sensor.Research supported by the Wellcome Trust

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min−1 flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

Previous studies have implicated annexins in regulating ion channels and in particular annexin A5 (AnxA5) in the traffic of the cystic fibrosis transmembrane conductance regulator (CFTR). In the present study, we further investigated the role of AnxA5 in regulating CFTR function and intracellular trafficking in both Xenopus oocytes and mammalian cells. Although we could confirm the previously reported CFTR/AnnxA5 interaction, we found that in oocytes AnxA5 inhibits CFTR-mediated whole-cell membrane conductance presumably by a mechanism independent of PDZ-binding domain at the C-terminus of CFTR but protein kinase C (PKC)-dependent and results from either endocytosis activation and/or exocytosis block. In contrast, in human cells, co-expression of AnxA5 augmented CFTR whole-cell currents, an effect that was independent of CFTR PDZ-binding domain. We conclude that annexin A5 has multiple effects on CFTR, so that the net effect observed is cell system-dependent. Nevertheless, both effects observed here are consistent with the described role of annexins forming scaffolding platforms at cell membranes, thus contributing to a decrease in their dynamics. Finally, we could not confirm that AnxA5 overexpression rescues traffic/function of the most frequent disease-causing mutant F508del-CFTR, thus concluding that AnxA5 is not a promising tool for correction of the F508del-CFTR defect.

The primary cause of cystic fibrosis (CF), the most frequent fatal genetic disease in Caucasians, is deletion of phenylalanine at position 508 (F508del), located in the first nucleotide‐binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR) protein. F508del‐CFTR is recognized by the endoplasmic reticulum quality control (ERQC), which targets it for proteasomal degradation, preventing this misfolded but partially functional Cl− channel from reaching the cell membrane. We recently proposed that the ERQC proceeds along several checkpoints, the first of which, utilizing the chaperone heat shock cognate 70 (Hsc70), is the major one directing F508del‐CFTR for proteolysis. Therefore, a detailed characterization of the interaction occurring between F508del‐CFTR and Hsc70 is critical to clarify the mechanism that senses misfolded F508del‐CFTR in vivo. Here, we determined by surface plasmon resonance that: (a) F508del‐murine (m)NBD1 binds Hsc70 with higher affinity (KD, 2.6 nm) than wild‐type (wt) mNBD1 (13.9 nm); (b) ATP and ADP dramatically reduce NBD1–Hsc70 binding; (c) the F508del mutation increases by approximately six‐fold the ATP concentration required to inhibit the NBD1–Hsc70 interaction (IC50; wt‐mNBD1, 19.7 μm ATP); and (d) the small molecule CFTR corrector 4a (C4a), but not VRT‐325 (V325; both rescuing F508del‐CFTR traffic), significantly reduces F508del‐mNBD1 binding to Hsc70, by ∼ 30%. Altogether, these results provide a novel, robust quantitative characterization of Hsc70–NBD1 binding, bringing detailed insights into the molecular basis of CF. Moreover, we show how this surface plasmon resonance assay helps to elucidate the mechanism of action of small corrective molecules, demonstrating its potential to validate additional therapeutic compounds for CF.Structured digital abstract MINT‐7265886: mNBD1 (uniprotkb:P26361) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by anti bait coimmunoprecipitation (MI:0006) MINT‐7265906, MINT‐7265964, MINT‐7265981, MINT‐7265951: mNBD1 (uniprotkb:P26361) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by surface plasmon resonance (MI:0107) MINT‐7265924, MINT‐7265939: hNBD1 (uniprotkb:P13569) binds (MI:0407) to Hsc70 (uniprotkb:P19120) by surface plasmon resonance (MI:0107) MINT‐7265996: Hsc70 (uniprotkb:P19120) binds (MI:0407) to Apo‐alpha‐lactalbumin (uniprotkb:P00711) by surface plasmon resonance (MI:0107)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl − channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1+2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PP i ) enhanced human, hmRD, and hmNBD1 CFTR Cl − currents, but not those of hmNBD2, hmNBD1+2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PP i .

The malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is associated with a wide spectrum of disease. In the search for modulators of CFTR, pharmaceutical agents have been identified that (i) act indirectly by regulating the protein kinases and phosphatases, which control CFTR, and (ii) interact directly with CFTR. Some agents modulate CFTR by altering the function of the nucleotide-binding domains (NBDs) that control channel gating, whereas others inhibit CFTR by preventing Cl− flow through the channel pore. Knowledge of CFTR modulators might lead to new understanding of the CFTR Cl− channel, its physiological role and malfunction in disease.

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.

Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed <--> open <--> blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.