Cystic Fibrosis Transmembrane Conductance Regulator Channel Function Research Articles

Molecular Structure of the Human CFTR Ion Channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.

S. aureus Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Therapeutics for the treatment of cystic fibrosis have recently progressed to include drugs that directly increase CFTR channel activity; however, the influence of bacterial virulence factors on the efficacy of these potentiators is not well understood. One secreted protein of particular interest is sphingomyelinase (SMase), a sphingomyelin-specific phosphodiesterase that generates the signaling lipid ceramide and has been shown previously to inhibit CFTR channel activity in Xenopus laevis oocytes and the immortalized Calu3 epithelial cell line. In this study we performed mechanistic experiments to better understand how bacterial sphingomyelinase impacts CFTR channel function and how that inhibition affects the efficacy of the clinically prescribed CFTR potentiator, Kalydeco (VX770). Our data shown that purified S. aureus SMase is a gating modifier of CFTR wherein channels are inhibited at the plasma membrane independent of the R-domain, the best understood regulatory domain of CFTR. We also found that inhibition occurs in channels that are isolated from the enzyme and does not occur in excised patches when SMase is backfilled into the pipette suggesting that inhibition occurs through a cytosolic signaling event. Consistent with the idea that SMase activity modulates CFTR gating at the membrane, we found that inhibition efficacy was dependent upon CFTR conformational state. Specifically, we found that mutations known to increase channel activity significantly decreased the rate of inhibition by SMase. Finally, we found evidence that inhibited channels cannot be rescued by applying VX770 suggesting that inhibited channels are effectively removed from the druggable pool. Taken together, our data suggest that S. aureus SMase is a gating modifier of CFTR that inhibits channel activity in a manner that renders VX770 ineffective.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

ABSTRACTCystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

SLC6A14 Enhances CFTR Channel Activity in the Cystic Fibrosis Affected Lung Epithelium

Cystic fibrosis (CF) is a common genetic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR protein functions apically on epithelial cells as an anion channel, helping to keep the surface hydrated. F508del-mutation in CFTR is the most common CF-causing mutation, which leads to decreased functional expression of the protein. However, patients homozygous for F508del mutation show a large variation in disease severity. To address this variation, previous genome-wide association studies have shown that SLC6A14 is a major modifier of CF disease severity. SLC6A14 is an electrogenic neutral/cationic amino-acid transporter expressed in colonic and lung epithelia. We examined the hypothesis that SLC6A14 has a positive impact on the functional expression of Wt and F508del-CFTR. Consistent with this hypothesis, we found that CFTR mediated anion-flux was enhanced by over-expression of SLC6A14 and addition of its transported substrate: arginine in a heterologous expression system. Similarly, in airway cell cultures endogenously expressing both SLC6A14 and CFTR, we observed that shRNA mediated knockdown of SLC6A14 reduced CFTR channel function. These findings could be translated to primary lung epithelial cultures derived from CF patients. The mechanism underlying the positive effect of SLC6A14 on CFTR function is currently unclear. It unlikely to be mediated via a direct interaction, since the two proteins could not be co-immunoprecipitated. However, it could be due to activation of intracellular signaling pathways leading to change in CFTR channel activity. Future studies will focus on the exploration of these pathways, and whether they affect the phosphorylation status of CFTR. Taken together, these findings give biologic insight to support that SLC6A14 is a modifier of CF disease severity. Hence, it can be used as an alternative drug target, especially in patients resistant to currently available therapies.

Modulation of Chloride Channel Functions by the Plant Lignan Compounds Kobusin and Eudesmin.

Plant lignans are diphenolic compounds widely present in vegetables, fruits, and grains. These compounds have been demonstrated to have protective effect against cancer, hypertension and diabetes. In the present study, we showed that two lignan compounds, kobusin and eudesmin, isolated from Magnoliae Flos, could modulate intestinal chloride transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs). The compounds activated CFTR channel function in both FRT cells and in HT-29 cells. The modulating effects of kobusin and eudesmin on the activity of CaCCgie (CaCC expressed in gastrointestinal epithelial cells) were also investigated, and the result showed that both compounds could stimulate CaCCgie-mediated short-circuit currents and the stimulation was synergistic with ATP. In ex vivo studies, both compounds activated CFTR and CaCCgie chloride channel activities in mouse colonic epithelia. Remarkably, the compounds showed inhibitory effects toward ANO1/CaCC-mediated short-circuit currents in ANO1/CaCC-expressing FRT cells, with IC50 values of 100 μM for kobusin and 200 μM for eudesmin. In charcoal transit study, both compounds mildly reduced gastrointestinal motility in mice. Taken together, these results revealed a new kind of activity displayed by the lignan compounds, one that is concerned with the modulation of chloride channel function.

Exploiting species differences to understand the CFTR Cl- channel.

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.

CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Altered cGMP Dynamics at the Plasma Membrane Contribute to Diarrhea in Ulcerative Colitis

Ulcerative colitis (UC) belongs to inflammatory bowel disorders, a group of gastrointestinal disorders that can produce serious recurring diarrhea in affected patients. The mechanism for UC- and inflammatory bowel disorder-associated diarrhea is not well understood. The cystic fibrosis transmembrane-conductance regulator (CFTR) chloride channel plays an important role in fluid and water transport across the intestinal mucosa. CFTR channel function is regulated in a compartmentalized manner through the formation of CFTR-containing macromolecular complexes at the plasma membrane. In this study, we demonstrate the involvement of a novel macromolecular signaling pathway that causes diarrhea in UC. We found that a nitric oxide-producing enzyme, inducible nitric oxide synthase (iNOS), is overexpressed under the plasma membrane and generates compartmentalized cGMP in gut epithelia in UC. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) bridges iNOS with CFTR, forming CFTR-NHERF2-iNOS macromolecular complexes that potentiate CFTR channel function via the nitric oxide-cGMP pathway under inflammatory conditions both invitro and invivo. Potential disruption of these complexes in Nherf2(-/-) mice may render them more resistant to CFTR-mediated secretory diarrhea than Nherf2(+/+) mice in murine colitis models. Our study provides insight into the mechanism of pathophysiologic occurrence of diarrhea in UC and suggests that targeting CFTR and CFTR-containing macromolecular complexes will ameliorate diarrheal symptoms and improve conditions associated with inflammatory bowel disorders.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

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(1),P(5)-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.

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not ΔF508 CFTR from thermal instability.

The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in ∼5% of cystic fibrosis patients worldwide. This amino acid substitution in NBD1 results in a CFTR chloride channel characterized by a severe gating defect that can be at least partially overcome in vitro by exposure to a CFTR potentiator. In contrast, the more common ΔF508 mutation is associated with a severe protein trafficking defect, as well as impaired channel function. Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on ΔF508 homozygotes. This difference in efficacy was not surprising in view of the established difference in the molecular phenotypes of the two mutant channels. Recently, however, it was shown that the structural defect introduced by the deletion of F508 is associated with the thermal instability of ΔF508 CFTR channel function in vitro. This additional mutant phenotype raised the possibility that the differences in the behavior of ΔF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 °C. We compared the thermal stability of G551D and ΔF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. G551D CFTR exhibited a thermal instability that was comparable to that of ΔF508 CFTR. G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas ΔF508 CFTR was not. These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature.

Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D‐CFTR

Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α-helix as G550 and R555. Both G550E and 4RK augmented strongly CFTR-mediated iodide efflux from BHK cells expressing G551D-CFTR. To learn how revertant mutations influence G551D-CFTR function, we studied protein processing and single-channel behaviour. Neither G550E nor 4RK altered the expression and maturation of G551D-CFTR protein. By contrast, both revertants had marked effects on G551D-CFTR channel gating, increasing strongly opening frequency, while 4RK also diminished noticeably the duration of channel openings. Because G551D-CFTR channel gating is ATP independent, we investigated whether revertant mutations restore ATP dependence to G551D-CFTR. Like wild-type CFTR, the activity of 4RK-G551D-CFTR varied with ATP concentration, suggesting that 4RK confers some ATP dependence on the G551D-CFTR channel. Thus, the revertant mutations G550E and 4RK alter the gating pattern and ATP dependence of G551D-CFTR without restoring single-channel activity to wild-type levels. Based on their impact on the CF mutants F508del and G551D, we conclude that G550E and 4RK have direct effects on CFTR structure, but that their action on CFTR processing and channel function is CF mutation specific.

Shank2 mutant mice display a hypersecretory response to cholera toxin

Shank2 is a PDZ (PSD-95/discs large/ZO-1)-based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2(-/-)) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2(+/+) and Shank2(-/-) mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short-circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR-Shank2-phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin-induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.

Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR channel structure and function, and of how CFTR activity is controlled by its local environment.

Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

Transcomplementation by a Truncation Mutant of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Enhances ΔF508 Processing through a Biomolecular Interaction

We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27-264 CFTR. Even though Δ27-264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27-264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27-264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27-264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27-264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.

Compartmentalized Regulation of CFTR Chloride Channel Function in Apical Epithelia

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is located primarily at the apical plasma membrane of polarized epithelial cells, plays a crucial role in transepithelial fluid homeostasis. Tight regulation of CFTR function is critical, as either hypofunctioning or hyperfunctioning of CFTR activity may result in life-threatening disorders. Accumulating evidence has suggested that the regulation of CFTR channel function is highly compartmentalized, i.e., CFTR function is tightly fine-tuned in localized microdomains, in which CFTR-containing multiprotein complexes play a critical role. A growing number of proteins has been reported to interact directly or indirectly with CFTR, implying that CFTR is coupled functionally and/or physically to a wide variety of interacting partners including ion channels, receptors, transporters, scaffolding proteins, enzyme molecules, signaling molecules, and effectors. In this review article, I summarized the most recent studies that characterize the compartmentalized regulation of CFTR function coupled to some interacting proteins such as receptors (i.e., adenosine receptor, beta adrenergic receptor, and lysophosphatidic acid receptor), transporters (i.e., cAMP efflux pump MRP4), and enzyme molecules (i.e., protein kinases, protein phosphatase, cAMP-specific phosphodiesterases, etc). These compartmentalized and dynamic interactions modulate not only the channel function, but also the localization and processing of CFTR protein within cells.

A Young Hispanic with c.1646G&gt;A Mutation Exhibits Severe Cystic Fibrosis Lung Disease: Is Ivacaftor an Option for Therapy?

A Young Hispanic with c.1646G&gt;A Mutation Exhibits Severe Cystic Fibrosis Lung Disease: Is Ivacaftor an Option for Therapy?

Macromolecular Complex of CFTR, NHERF2 and iNOS at the Plasma Membrane Contributes to Diarrhea in IBD

Macromolecular complex formation is a common theme in regulation of protein function. The cystic fibrosis transmembrane conductance regulator (CFTR), a cGMP/cAMP‐regulated chloride (Cl−) channel, is a well‐known hub protein involved in multiple protein complexes. In this study, we find that a signaling macromolecular complex of CFTR, Na+/H+ exchanger regulatory factor 2 (NHERF2), and inducible Nitric Oxide Synthase (iNOS) is formed at the plasma membrane in Inflammatory Bowel disease (IBD). We demonstrated the protein interactions using biochemical methods (e.g., protein pairwise binding assay, macromolecular complex assembly assay, and co‐immunoprecipitation) and in vitro methods for studying protein interaction in live cell (e.g., FRET). We conclude based on our in vitro (polarized colonic epithelial monolayers) and in vivo (DSS‐induced colitis in mice) functional studies that the interaction is integrative to CFTR dependent diarrhea in IBD. To summarize our findings: (a) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain‐dependent manner. (b) Upregulation of iNOS increased CFTR channel function by at least two folds in in vivo and in vitro models of inflammation. (c). Diarrhea was attenuated in Nherf2−/− mice compared to Nherf2+/+mice in DSS‐induced colitis model which simulates IBD. Taken together, our results demonstrate that the macromolecular complex of CFTR, NHERF2 and iNOS play important roles in the pathogenic process of diarrhea in IBD. Our study will not only help understand the molecular mechanisms underlying the pathogenic process of diarrhea in IBD, but help to identify novel therapeutic targets and agents for IBD therapy.

Recent Advances And New Perspectives In Targeting CFTR For Therapy Of Cystic Fibrosis And Enterotoxin-Induced Secretory Diarrheas

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical surfaces of epithelial cells lining airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with an altered channel function of CFTR. Cystic fibrosis (CF) is caused by the loss or dysfunction of CFTR-channel activity resulting from the mutations on the gene; whereas enterotoxin-induced secretory diarrheas are caused by the hyperactivation of CFTR channel function. CFTR is a validated target for drug development to treat these diseases. Significant progress has been made in developing CFTR modulator therapy by means of high-throughput screening followed by hit-to-lead optimization. Several oral administrated investigational drugs are currently being evaluated in clinical trials for CF. Also importantly, new ideas and methodologies are emerging. Targeting CFTR-containing macromolecular complexes is one such novel approach.

Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.