Gating Defects Research Articles

Pharmacological Correction of Gating Defects in the Voltage-Gated Cav2.1 Ca2+ Channel due to a Familial Hemiplegic Migraine Mutation

Pharmacological Correction of Gating Defects in the Voltage-Gated Cav2.1 Ca2+ Channel due to a Familial Hemiplegic Migraine Mutation

Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Highlights from the Literature

Highlights from the Literature

P.D1690N Nav1.5 rescues p.G1748D mutation gating defects in a compound heterozygous Brugada syndrome patient

We identified 2 compound heterozygous mutations (p.D1690N and p.G1748D) in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) in a proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele encoding the p.D1690N mutation, the p.H558R polymorphism was also detected. The purpose of this study was to analyze the functional properties of the mutated channels as well as the putative modulator effects produced by the presence of the polymorphism. Wild-type and mutated human Nav1.5 channels were expressed in Chinese hamster ovary cells and recorded using whole-cell patch-clamp technique. Separately, both p.D1690N and p.G1748D mutations produced a marked reduction in peak Na(+) current density (>80%), mainly due to their limited trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly affected channel gating. Both p.D1690N and p.G1748D produced a marked dominant negative effect when cotransfected with either wild-type or p.H558R channels. Conversely, p.H558R was able to rescue defective trafficking of p.D1690N channels toward the membrane when both polymorphism and mutation were in the same construct. Surprisingly, cotransfection with p.D1690N, either alone or together with the polymorphism (p.H558R-D1690N), completely restored the profound gating defects exhibited by p.G1748D channels but only slightly rescued their trafficking. Our results add further support to the hypothesis that Nav1.5 subunits interact among them before trafficking toward the membrane and suggest that a missense mutation can "rescue" the defective gating produced by another missense mutation.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

A new model of Pde4d deficiency: genetic knock‐down of PDE4D enzyme in rats produces an antidepressant phenotype without spatial cognitive effects

Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes composed of 12 families. The Pde4 family has been implicated in depression and cognition, and PDE4 inhibitors have been evaluated as antidepressants and possible cognitive enhancers. Pde4d(-/-) mice show an antidepressant phenotype and learning enhancement on some tests, but not others as do mice treated with PDE4 inhibitors. Here, we report for the first time the behavioral phenotype of a new Pde4d knock-down (KD) rat model of PDE4D deficiency. Consistent with other data on PDE4D deficiency, Pde4d KD rats showed depression resistance in the Porsolt forced swim test and hyperreactivity of the acoustic startle response with no differential response on prepulse inhibition, suggesting no sensorimotor gating defect. Pde4d KD rats also exhibited a small exploratory activity reduction but no difference following habituation, and no enhanced spatial learning or reference memory in the Morris water maze. A selective improvement in route-based learning in the Cincinnati water maze was seen as well as enhanced contextual and cued fear conditioning and a more rapid rate of cued extinction from their higher freezing level that declined to wild-type (WT) levels only after ∼20 extinction trials. The rat model confirms Pde4d's role in depression but not in spatial learning or memory enhancement and shows for the first time higher fear conditioning and altered extinction compared with controls. The new model provides a tool by which to better understand the role of PDE4D in neuropsychiatric disorders and for the development of alternate treatment approaches.

NMD‐deficient Upf3b‐null mice display behavioral and neuropathological defects

To address the physiological role of nonsense‐mediated RNA decay (NMD), we generated mice with a null mutation in the Upf3b NMD gene. While these Upf3b‐null mice are viable, fertile, and grossly normal, a battery of behavioral tests revealed they exhibit a specific defect in sensorimotor gating. This defect is commonly displayed in Schizophrenia patients, a subset of whom have recently been shown by several groups to have mutations in the human UPF3B gene. Neuropathological examination of the frontal cortex, the site responsible for sensorimotor gating, revealed decreased dendritic spine density and mature dendritic spines in pyramidal cells. Intriguingly, such defects are also observed in schizophrenic and autistic patients. To understand the underlying mechanisms by which UPF3B acts in this circuit, we performed exon‐array analysis of the frontal cortex of Upf3b‐null mice. This revealed dysregulation of several mRNAs transcribed from genes mutated in intellectual disability patients, most of which encode proteins that function in sensory perception and cognition. NMD is likely to have a direct role in regulating many of these transcripts, as most have “NMD‐inducing features.” We propose that Upf3b‐null mice are a useful model system to understand the underlying mechanism of sensorimotor gating and how it is disrupted in Schizophrenia patients.This study was supported by the NIH.

Ivacaftor potentiation of multiple CFTR channels with gating mutations

BackgroundThe investigational CFTR potentiator ivacaftor (VX-770) increased CFTR channel activity and improved lung function in subjects with CF who have the G551D CFTR gating mutation. The aim of this in vitro study was to determine whether ivacaftor potentiates mutant CFTR with gating defects caused by other CFTR gating mutations. MethodsThe effects of ivacaftor on CFTR channel open probability and chloride transport were tested in electrophysiological studies using Fischer rat thyroid (FRT) cells expressing different CFTR gating mutations. ResultsIvacaftor potentiated multiple mutant CFTR forms with defects in CFTR channel gating. These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations. ConclusionThese in vitro data suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support investigation of the potential clinical benefit of ivacaftor in CF patients who have CFTR gating mutations beyond G551D.

Mapping GABAA Receptor Single Nucleotide Polymorphisms (SNPs) Linked to Epilepsy: Insights into the Receptor Gating and Assembly

Missense polymorphisms or variants that affect function and/or expression of GABAA receptors (GABARs) have been associated with idiopathic generalized epilepsies (IGEs) in GABRA1, GABRA6, GABRB3, GABRG2 and GABRD genes. Three separate IGE-associated mutations were identified in GABAR subunits (β3(G32R), α6(Q237R) and γ2(K328M)), which represent a large range of GABARs in the nervous system. We sought to investigate the contributions of these mutations into the assembly and function of GABARs. Homology modeling suggested that G32R is located within the N-terminal α-helix β3 subunit domain, and Q237R and K328M are located within the pre-M1 segment of the N-terminal α6 subunit domain and the M2-M3 loop of the N-terminal γ2 subunit domain, respectively. We studied gating properties and surface expression of wild type (wt) α1β3γ2, α6β2γ2, α1β2γ2 and mutant α1β3(G32R)γ2, α6(Q237R)β2γ2, α1β2γ2(K328M) receptors expressed in HEK293T cells. We found that the mutations share common gating defects, but distinctive trafficking defects. Thus, mutant β3(G32R) subunits displayed a mixed profile, causing both gating and trafficking defects of α1β3γ2 receptors, whereas mutant α6(Q237R) and γ2(K328M) subunits caused exclusive channel gating defects of α6β2γ2 and α1β2γ2 receptors. Unexpected, homology modeling predicted that the β3(G32R) mutation affects a salt bridge across the γ2/β3 subunit interface, which underlies an assembly motif reported to be essential for inter-subunit interactions in assembled receptors. In contrast, α6(Q237R) and γ2(K328M) subunit mutations are predicted to interact with residues in Pre-M1 domain, M2-M3 loop and Cys-loop of α6 and γ2 subunits that are critical for desensitization-deactivation coupling of GABARs.NIH funding NS33300 to RLM.

Use the subthreshold-current technique to separate radiation induced defects in gate controlled lateral pnp bipolar transistors

In this paper, we design and fabricate a new test structure of bipolar device. A gate is deposited on the oxide layer covering the base region of normal lateral pnp bipolar transistor. The characteristic of drain current (collector current) versus the gate voltage is recorded by sweeping the voltage applied to the gate, then the subthreshold-current technique is used to separate the radiation induced oxide trapped charges and interface traps in the gate controlled lateral pnp bipolar transistor during 60Co- irradiation. The test structure and the measurement of the bipolar transistor used in the experiment are introduced in detail in this paper.

Human–mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-ΔF508 processing and alter its gating defect

The ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the most common cause of cystic fibrosis. The mutation disrupts biosynthetic processing, reduces channel opening rate, and decreases protein lifetime. In contrast to human CFTR (hCFTR)-ΔF508, mouse CFTR-ΔF508 is partially processed to the cell surface, although it exhibits a functional defect similar to hCFTR-ΔF508. To explore ΔF508 abnormalities, we generated human-mouse chimeric channels. Substituting mouse nucleotide-binding domain-1 (mNBD1) into hCFTR partially rescued the ΔF508-induced maturation defect, and substituting mouse membrane-spanning domain-2 or its intracellular loops (ICLs) into hCFTR prevented further ΔF508-induced gating defects. The protective effect of the mouse ICLs was reverted by inserting mouse NBDs. Our results indicate that the ΔF508 mutation affects maturation and gating via distinct regions of the protein; maturation of CFTR-ΔF508 depends on NBD1, and the ΔF508-induced gating defect depends on the interaction between the membrane-spanning domain-2 ICLs and the NBDs. These appear to be distinct processes, because none of the chimeras repaired both defects. This distinction was exemplified by the I539T mutation, which improved CFTR-ΔF508 processing but worsened the gating defect. Our results, together with previous studies, suggest that many different NBD1 modifications improve CFTR-ΔF508 maturation and that the effect of modifications can be additive. Thus, it might be possible to enhance processing by targeting several different regions of the domain or by targeting a network of CFTR-associated proteins. Because no one modification corrected both maturation and gating, perhaps more than a single agent will be required to correct all CFTR-ΔF508 defects.

Reduced Light Response of Neuronal Firing Activity in the Suprachiasmatic Nucleus and Optic Nerve of Cryptochrome-Deficient Mice

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day–night difference was not observed in Cry-deficient mice. In addition, we observed day–night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals.

Targeting F508del-CFTR to develop rational new therapies for cystic fibrosis

The mutation F508del is the commonest cause of the genetic disease cystic fibrosis (CF). CF disrupts the function of many organs in the body, most notably the lungs, by perturbing salt and water transport across epithelial surfaces. F508del causes harm in two principal ways. First, the mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Second, F508del perturbs the Cl(-) channel function of CFTR by disrupting channel gating. Here, we discuss the development of rational new therapies for CF that target F508del-CFTR. We highlight how structural studies provide new insight into the role of F508 in the regulation of channel gating by cycles of ATP binding and hydrolysis. We emphasize the use of high-throughput screening to identify lead compounds for therapy development. These compounds include CFTR correctors that restore the expression of F508del-CFTR at the apical membrane of epithelial cells and CFTR potentiators that rescue the F508del-CFTR gating defect. Initial results from clinical trials of CFTR correctors and potentiators augur well for the development of small molecule therapies that target the root cause of CF: mutations in CFTR.

The most common cystic fibrosis‐associated mutation destabilizes the dimeric state of the nucleotide‐binding domains of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), can increase the open probability (Po) to ∼0.7, implying that the gating defect of ΔF508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PPi), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PPi-induced locked-open time and the ATP/P-ATP ligand exchange time of ΔF508-CFTR channels are dramatically shortened, suggesting that the ΔF508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of ΔF508-CFTR, namely the solubilizing mutation F494N/Q637R and ΔRI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with ΔF508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of ΔF508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for ΔF508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.

A Single Pulse Charge Pumping Technique for Fast Measurements of Interface States

Characterizing interface states is a key task, and it typically takes seconds when conventional techniques, such as charge pumping (CP), are used. The stress-induced degradation can recover substantially during this time, and there is a need to improve the measurement speed. The central task of this work is to reduce the measurement time for interface states from seconds to microseconds to minimize the recovery. A fast single pulse CP (SPCP) technique is developed. By exploring the differences in the transient currents corresponding to the two edges of the gate pulse, the net charges pumped into devices can be obtained, and their saturation level is used to evaluate interface states. Unlike the conventional CP (CCP) method, the contribution of currents during the plateaus of gate pulse is excluded for SPCP, making it less vulnerable to the interferences of gate leakage and defects within dielectrics. For the first time, the SPCP allows the recovery of interface states being monitored with a time resolution in microseconds. The results show that the recovery of stress-induced interface states is substantial within 100 μs, which would be missed if the CCP were used.

Metastability mechanisms in thin film transistors quantitatively resolved using post-stress relaxation of threshold voltage

A new approach is presented to resolve bias-induced metastability mechanisms in hydrogenated amorphous silicon (a-Si:H) thin film transistors (TFTs). The post stress relaxation of threshold voltage (VT) was employed to quantitatively distinguish between the charge trapping process in gate dielectric and defect state creation in active layer of transistor. The kinetics of the charge de-trapping from the SiN traps is analytically modeled and a Gaussian distribution of gap states is extracted for the SiN. Indeed, the relaxation in VT is in good agreement with the theory underlying the kinetics of charge de-trapping from gate dielectric. For the TFTs used in this work, the charge trapping in the SiN gate dielectric is shown to be the dominant metastability mechanism even at bias stress levels as low as 10 V.

Dual Activity of Aminoarylthiazoles on the Trafficking and Gating Defects of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Caused by Cystic Fibrosis Mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.

Curcumin and genistein additively potentiate G551D-CFTR

Background The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. Methods We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. Results Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~ 50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. Conclusions The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

Combating Cystic Fibrosis: In Search for CF Transmembrane Conductance Regulator (CFTR) Modulators

Open sesame! Compounds that correct the folding, trafficking and gating defects in cystic fibrosis transmembrane conductance regulator (CFTR), the main protein implicated in cystic fibrosis (CF), are of great interest as potential therapeutic agents for this lethal disease. The development of CFTR modulators, highlighting the main challenges in the field and advocating the application of rational rather than brute-force methods, is the focus of this Minireview.

A common SCN5A polymorphism modulates the biophysical defects of SCN5A mutations

Defects in the cardiac sodium channel gene, SCN5A, can cause a broad spectrum of inherited arrhythmia syndromes. After genotyping of a proband who presented with syncope, the SCN5A mutant P2006A and the common polymorphism H558R were identified. The main objective of this study was to determine whether the SCN5A-H558R polymorphism could modify the defective gating kinetics observed in the P2006A mutation and therefore explain why this gain-of-function mutation has been identified in control populations. Mutations were engineered using site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. Whole-cell sodium currents were measured at room temperature using the whole-cell patch-clamp technique. In HEK293 cells, P2006A displayed biophysical defects typically associated with long QT syndrome by increasing persistent sodium current, producing a depolarizing shift in voltage dependence of inactivation, and hastening recovery from inactivation. Interestingly, when coexpressed either on the same or different genes, P2006A and H558R displayed currents that behaved like wild type (WT). We also investigated whether H558R can modulate the gating defects of other SCN5A mutations. The H558R polymorphism also restored the gating defects of the SCN5A mutation V1951L to the WT level. Our results suggest that H558R might play an important role in stabilization of channel fast inactivation and may provide a plausible explanation as to why the P2006A gain-of-function mutation has been identified in control populations. Our results also suggest that the SCN5A polymorphism H558R might be a disease-modifying gene.