Gating Defects Research Articles

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

Reliability of Highly Stable Amorphous-Silicon Thin-Film Transistors Under Low Gate-Field Stress—Part II: Optimization of Fabrication Conditions and Gate Voltage Dependence

Part I of this paper introduced a two-stage model for reliability characterization and lifetime prediction of amorphous-silicon thin-film transistors (a-Si TFTs) under low gate-field stress that includes both charge trapping in the silicon nitride (SiNx) gate dielectric and defect generation in the a-Si channel. In part II, the model is used to experimentally reduce the drain current instability under room temperature operation of a-Si TFTs under a prolonged gate bias of 5 V. Deposition conditions for the SiNx gate insulator and the a-Si channel layer were varied, and TFTs were fabricated with all reactive-ion-etch steps, or with all wet-etch steps. The stability of the a-Si channel also depends on the deposition conditions for the underlying SiNx gate insulator, and TFTs made with wet etching are more stable than TFTs made with reactive ion etching. Combining the various improvements raised the extrapolated 50% lifetime of the drain current of back-channel-passivated dry-etched TFTs under continuous operation in saturation at 20 °C with $V_{\mathrm{GS}}=\text{5} \text{V}$ from $\text{3}\times \text{10}^{4} \text{s}$ (9.2 h) to $\text{4}\times \text{10}^{7} \text{s}$ (1.4 years). We also extend the model, so that parameters from the degradation at one gate voltage can be used to estimate the degradation at other low gate voltages.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.

Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.

HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms

The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

Effects of interface and bulk properties of gate-dielectric on the performance and stability of hydrogenated amorphous silicon thin-film transistors

In order to investigate the effects of interface and bulk properties of gate insulator on the threshold voltage (Vth) and the gate-bias induced instability of hydrogenated amorphous silicon thin-film transistors (a-Si:H TFTs), four kinds of TFT structures were fabricated with SiNx and SiOx insulators stacked to make different combinations of the bulk and interface in the gate-dielectric layers. It was found that the Vth and the stability are independently controlled by tuning stoichiometry and thickness of the SiOx insertion layer between a-Si:H and SiNx. In TFTs with SiOx insertion layer of 50 nm thickness, on increasing oxygen/silicon (O/Si = x) ratio from 1.7 to 1.9, Vth increased from 0 V to 9 V. In these TFTs with a relatively thick SiOx insertion layer, positive Vth shift with negative bias stress was observed, confirmed to be due to defect creation in a-Si:H with the thermalization barrier energy of 0.83 eV. On reducing the thickness of the SiOx insertion layer down to approximately 1 nm, thin enough for hole injection through SiOx by tunneling effect, stable operation was obtained while keeping the high Vth value under negative stress bias. These results are consistently explained as follows: (1) the high value for Vth is caused by the dipole generated at the interface between a-Si:H and SiOx; and (2) two causes for Vth shift, charge injection to the gate insulator and defect creation in a-Si:H, are mutually related to each other through the “effective bias stress,” Vbseff = Vbs – ΔVfb (Vbs: applied bias stress and ΔVfb: flat band voltage shift due to the charge injection). It was experimentally confirmed that there should be an optimum thickness of SiOx insertion layer of approximately 1 nm with stable high Vth, where enhanced injection increases ΔVfb, reduces Vbseff to reduce defect creation, and totally minimizes Vth shift.

Lumacaftor alone and combined with ivacaftor: preclinical and clinical trial experience of F508del CFTR correction

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR), leading to significant morbidity and mortality. CFTR is a chloride and bicarbonate channel at the epithelial cell membrane. The most common CFTR mutation is F508del, resulting in minimal CFTR at the plasma membrane. Current disease management is supportive, whereas an ultimate goal is to develop therapies to restore CFTR activity. We summarize experience with lumacaftor, a small molecule that increases F508del-CFTR levels at the plasma membrane. Lumacaftor in combination with ivacaftor, a modulator of CFTR gating defects, improves clinical outcome measures in patients homozygous for the F508del mutation. Lumacaftor represents a significant advancement in the treatment of biochemical abnormalities in CF. Further development of CFTR modulators will improve upon current therapies, although it remains unclear whether this approach will provide therapies for all CFTR mutations.

Combined gating and trafficking defect in Kv11.1 manifests as a malignant long QT syndrome phenotype in a large Danish p.F29L founder family

Background. Congenital long QT syndrome (LQTS) is a hereditary cardiac channelopathy characterized by delayed ventricular repolarization, syncope, torsades de pointes and sudden cardiac death. Thirty-three members of five apparently ‘unrelated’ Danish families carry the KCNH2:c.87C> A; p.F29L founder mutation. Methods and Results. Linkage disequilibrium mapping with microsatellites around KCNH2 enabled us to estimate the age of the founder mutation to be approximately 22 generations, corresponding to around 550 years. Neighbouring-Joining analysis disclosed one early and three later nodes. The median QTc time of the carriers was 490 ms (range: 415–589 ms) and no difference was seen between the different branches of the family. The mutation is malignant with a penetrance of 73%. Ten F29L carriers received implantable defibrillators (ICDs) (median age at implant 20 years), and of those four individuals experienced eight appropriate shocks. Patch-clamp analysis in HEK 293 cells, performed at 34°C disclosed a loss-of-function phenotype with fast deactivation, reduced steady-state inactivation current density and a positive voltage shift in inactivation. Western blotting of HEK 293 cells transfected with KCNH2:WT and KCNH2:c.87C> A revealed a reduced fraction of fully glycosylated hERG:p.F29L suggesting that this mutation results in defective trafficking. Conclusion. The altered channel gating kinetics in combination with defective trafficking of mutated channels is expected to result in reduced repolarizing current density and, thus, a LQTS phenotype.

Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization

Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

Murine and human CFTR exhibit different sensitivities to CFTR potentiators.

Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as "Kalydeco." Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by ∼13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators.

Performance scaling of the quantum Fourier transform with defective rotation gates

Addressing Landauer's question concerning the influence of static gate defects on quantum information processor performance, we investigate analytically and numerically the case of the quantum Fourier transform (QFT) with defective controlled rotation (CROT) gates. Two types of defects are studied, separately and in combination: systematic and random. Analytical scaling laws of QFT performance are derived with respect to the number of qubits $n$, the size $\delta$ of systematic defects, and the size $\epsilon$ of random defects. The analytical results are in excellent agreement with numerical simulations. In addition, we present an unexpected result: The performance of the defective QFT does not deteriorate with increasing $n$, but approaches a constant that scales in $\epsilon$. We derive an analytical formula that accurately reproduces the $\epsilon$ scaling of the performance plateaus. Overall, we observe that the CROT gates may exhibit static and random defects of the order of 30\% and larger, and still result in satisfactory QFT performance. Thus we answer Landauer's question in the case of the QFT: far from being lethal, the QFT can tolerate tremendous static gate defects and still perform its function. The extraordinary robustness of the QFT with respect to static gate defects displayed in our numerical and analytical calculations should be a welcome boon for laboratory and industrial realizations of quantum circuitry.

Synthesis and structure–activity relationship of aminoarylthiazole derivatives as correctors of the chloride transport defect in cystic fibrosis

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expressing F508del-CFTR using a functional assay. A dual active compound, AAT-4a, characterized by improved efficacy and marked synergy when combined with the corrector VX-809 has been identified. Moreover, by computational methods, a possible binding site for AATs in nucleotide binding domain NBD1 has been detected. These results will direct the synthesis of new analogues with possibly improved activity.

Improvement of Hepatic Steatosis in Cystic Fibrosis With Ivacaftor Therapy

Treatment of liver disease, including hepatic steatosis, in patients with cystic fibrosis (CF) is limited. With the development of ivacaftor, which corrects the gating defect of the CF transmembrane regulator channel, there is a potential new therapy available for this subgroup of the CF patient population. We present an adolescent with CF who had significant improvement in hepatic steatosis with ivacaftor treatment while hypothesizing on a mechanism of why it occurred.

Molecular Basis for Fe(III)-Independent Curcumin Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Activity.

Curcumin potentiates the phosphorylation-dependent activity of human cystic fibrosis transmembrane conductance regulator (CFTR) in Fe(3+)-dependent and Fe(3+)-independent manners. Although the Fe(3+)-dependent curcumin potentiation results from removal of endogenous inhibitory Fe(3+) at the interface of the regulatory (R) domain and intracellular loop (ICL) 3, the molecular mechanism of Fe(3+)-independent curcumin potentiation is still unknown. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of a highly conserved ICL1/ICL4 interface and a stimulatory phosphorylation site S795 or S813 at the C-terminus of the R domain in this potentiation pathway was investigated. The results showed that highly conserved aromatic and positively charged residues at the ICL1/ICL4 interface and phosphorylation site S813 were sensitive to curcumin regardless of whether Fe(3+) and nucleotide-binding domain 2 were removed. More importantly, spontaneous disulfide cross-linking between curcumin-sensitive ICL1 and S795 was observed to be enough to promote channel opening as curcumin did. Therefore, the phosphorylated R domain, once released from ICL3, may function as a length- and gating-regulatory cross-linker between two transmembrane domains to promote the stimulatory interactions between the R domain and the ICL1/ICL4 interface. Curcumin may potentiate CFTR activity not only by removing inhibitory Fe(3+) to release the R domain from ICL3 but also by stabilizing the stimulatory R-ICL1/ICL4 interactions. Therefore, the future potentiators with two linked aromatic rings could utilize this potentiation pathway to bypass the need for ATP to rescue the activity of cystic fibrosis mutants with an ATP-dependent gating defect.

Targeting Disabled‐2 to Stabilize ΔF508‐CFTR

The fatal genetic disease Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, ΔF508‐CFTR, negatively impacts the processing, gating, and stability of CFTR. Existing combination therapies elicit only modest benefit in ΔF508‐CFTR patients. Potentiators and correctors address the gating and processing defects of ΔF508‐CFTR, respectively, but do not specifically target its stability. Thus, by targeting additional aspects of the mutant CFTR life cycle we may significantly improve CF patient prognosis.
 Here, we present evidence for a new class of stabilizers that target CFTR half‐life. The adaptor protein Disabled‐2 (Dab2), is an essential mediator of CFTR endocytosis. The Dab2 Dab homology (DH) domain is required for Dab2 to co‐assemble in complex with CFTR, and our lab has shown that DH peptide inhibitors enhance ΔF508‐CFTR function alone and even more so when combined with correctors and potentiators. We have now used an engineered peptide with high‐affinity for the DH domain as a reporter in a fluorescence anisotropy based high‐throughput screen to identify candidate small‐molecule inhibitors. FRET and NMR are used to further characterize the intermolecular binding between the DH domain and potential stabilizers.

Neurogenesis and sensorimotor gating: bridging a microphenotype and an endophenotype.

Human genetic data on psychiatric disorders repeatedly demonstrate the involvement of various genes that are associated with neural development and neurogenesis. Neurogenesis is a biological process that is critical in brain development and continues throughout life. Neurogenesis is a multi-step process starting from the division of neural stem cells/progenitor cells, leading to self-renewal and simultaneously to the production of lineage-committed cells, including neurons and glial cells. Minor defects in the neurogenesis process, such as production of fewer new neurons and malformation of neural circuits, could represent phenotypes of psychiatric disorders at molecular and cellular levels in animal models (here termed as "microphenotypes"). However, microphenotypes are not easily used as biomarkers. We have focused on a physiological condition, sensorimotor gating deficits, that can be scored by a prepulse inhibition (PPI) test. Impaired PPI is considered to be one of the compelling endophenotypes (biological markers) of mental disorders such as schizophrenia, autism, and other neurodevelopmental disorders. Because the neural circuit for PPI involves the hippocampus, a unique brain region where neurogenesis occurs postnatally, we hypothesize that an impairment of preadolescent neurogenesis is critical for the onset of sensorimotor gating defects. To test this hypothesis, we investigated a critical period of neurogenesis that can affect PPI. In this paradigm, we introduced an enriched environment to restore neurogenesis, thereby recovering PPI deficits in mice. We noted impairments in the maturation of newborn neurons in the hippocampal dentate gyrus (DG) and GABAergic neurons in the hippocampus, which could be considered as microphenotypes associated with PPI defects. More precise genetically controlled neurogenesis models (with precise time points or periods) are needed to be studied in further investigation to support our hypothesis.

Improved Clinical and Radiographic Outcomes After Treatment With Ivacaftor in a Young Adult With Cystic Fibrosis With the P67L CFTR Mutation

The underlying cause of cystic fibrosis (CF) is the loss of epithelial chloride and bicarbonate transport due to mutations in the CF transmembrane conductance regulator (CFTR) gene encoding the CFTR protein. Ivacaftor is a gene-specific CFTR potentiator that augments in vivo chloride transport in CFTR mutations affecting channel gating. Originally approved for the G511D CFTR mutation, ivacaftor is now approved for eight additional alleles exhibiting gating defects and has also been tested in R117H, a CFTR mutation with residual function that exhibits abnormal gating. P67L is a class 4 conductance (nongating) mutation exhibiting residual CFTR function. We report marked clinical improvement, normalization of spirometry, and dramatic reduction in radiographic structural airway changes after > 1 year of treatment with ivacaftor in a young adult with the compound heterozygous genotype P67L/F508del CFTR. The case suggests that ivacaftor may have a potential benefit for patients with CF with nongating mutations.

On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process.

PKA-Dependent Potentiation Mechanisms of Human CFTR Activity

Both curcumin and VX-770 potentiate the channel activity of human CFTR (hCFTR) and two most common cystic fibrosis (CF) mutants G551D and F508delta in an ATP-independent but PKA-dependent manner. The underlying molecular mechanisms are unclear. Herein, HEK-293T cells cultured in a Fe3+-containing medium were transiently transfected with hCFTR constructs and curcumin with well-known chemical nature was employed as a template to explore PKA-dependent potentiation mechanisms of hCFTR activity. The results showed that curcumin potentiation of Fe3+-sensitive hCFTR activity was partially weakened by Fe3+-insensitive mutations at the interface of the R domain and intracellular loop (ICL) 3 and completely suppressed by sufficient Fe3+. Thus, release of the inhibitory Fe3+-bound R domain from ICL3 by curcumin may be critical for curcumin potentiation. Further study indicated that curcumin potentiation was significantly prohibited by a missense alanine mutation of F157, Y161 or K166 from ICL1, or R1066, F1074 or F1078 from ICL4, or S795 or S813 from the R domain with or without the involvement of nucleotide-binding domain 2 (NBD2). More importantly, curcumin potentiation was also suppressed by the R811A/S813D or Y808A/S813D mutation and disulfide crosslinking of K162C to S795C enhanced channel opening. Therefore, the phosphorylated R domain may function as a length- and gating-regulatory cross-linker between two transmembrane domains (TMD1 and TMD2). Curcumin may potentiate hCFTR activity by stabilizing the stimulatory ICL1/ICL4-R interactions that promote channel opening by pulling all ICLs together and thus triggering a gating inward-to-outward reorientation of TMDs. Possible chemical interactions may involve cation-π interactions, π-π interactions and hydrogen bonding. Taken together, both release of the R domain from ICL3 and the stimulatory R-ICL1/ICL4 interactions may be necessary for PKA-dependent hCFTR activation and potentiation. These findings may help optimize the potentiators for treating those CF mutants with an ATP-dependent gating defect.

Tunable plasmonic band gap and defect mode in one-dimensional photonic crystal covered with graphene

We study the transmission characteristics of surface plasmons at the interface of a one-dimensional photonic crystal (1DPC) and a monolayer graphene sheet. The composite structure with a plasmonic band gap (PGB) was obtained by covering a graphene sheet on the lateral side of the 1DPC. Defect modes in these structures appeared when defect layers were inserted in the 1DPC. On the condition that the chemical potential of graphene is much larger than , the PBG and defect mode shift to higher frequencies as the chemical potential increases. As the carrier density in graphene can be controlled by the gate voltage, both the PBG and defect mode can be easily tuned by the chemical potential, which is determined by the carrier density.