Deactivation Properties Research Articles

EFFECT OF MICROSTRUCTURES ON MASS TRANSFER INSIDE A HIERARCHICALLY STRUCTURED POROUS CATALYST

In this paper, the pore network model to investigate the reaction-diffusion process in the hierarchically structured porous catalyst particle is extended to consider the phenomenon of deactivation by coking. In this framework, the interaction of internal particle pore structure and mass transfer under the condition of coke deposition is examined. Then, the effect of structural features, namely macroporosity and pore size ratio, the deactivation properties, the maximum loading of coke, as well as the transport properties, the pore Damköhler number on the net reaction rate, and deactivation of the particle have been investigated. Three deactivation mechanisms are accounted for, namely, the site coverage, the pore narrowing, and the pore blockage. It is found that the deactivation of the catalyst particle can be divided into two conditions: the kinetic deactivation and the structural deactivation. It is shown that depending on the Damköhler number, increasing the macroporosity does not necessarily improve the reactivity and deactivation resistance of the catalyst. The key finding of this work is to demonstrate and quantify how changing the typical fresh catalyst microstructure into a hierarchical one influences the reactivity and deactivation.

MCM-22 zeolite-based system to produce jet fuel from the 1-hexene oligomerization

The oligomerization of light olefins is an alternative for generating clean liquid fuels. In this study, the MCM-22 zeolite featuring a partially disordered layered structure with framework combined 10 membered ring (10 MR) was discussed for 1-hexene oligomerization. MCM-22 and Y zeolite were compared to investigate the influences of the framework and acidity strength on the activity and yield in the 1-hexene oligomerization. The results indicated that the MCM-22 zeolite was an efficient catalyst for oligomerization compared to Y zeolite. The MCM-22 zeolite was characterized in detail by NMR, Py-FTIR, and TG analyses. The catalytic performance and deactivation properties of MCM-22 zeolite in the oligomerization reaction of 1-hexene were investigated. Structure–activity relationships were established through the kinetic study. Although the density of strong acid sites was essential for the oligomerization of 1-hexene, the presence of low pressure was also necessary for the formation of dimers. Thus, MCM-22 zeolite demonstrates a substantial improvement in initial conversion and catalyst lifespan for 1-hexene oligomerization.

Shisa7-Dependent Regulation of GABAA Receptor Single-Channel Gating Kinetics.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABAAR-associated transmembrane proteins. We previously identified Shisa7 as a GABAAR auxiliary subunit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABAAR single-channel properties has yet to be determined. Here, we performed single-channel recordings of α2β3γ2L GABAARs cotransfected with Shisa7 in HEK293T cells and found that while Shisa7 does not change channel slope conductance, it reduced the frequency of receptor openings. Importantly, Shisa7 modulates GABAAR gating by decreasing the duration and open probability within bursts. Through kinetic analysis of individual dwell time components, activation modeling, and macroscopic simulations, we demonstrate that Shisa7 accelerates GABAAR deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABAAR gating and reveal for the first time that GABAAR single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission.SIGNIFICANCE STATEMENT Although GABAA receptor (GABAAR) single-channel properties are largely determined by pore-forming subunits, it remains unknown whether they are also controlled by GABAAR-associated transmembrane proteins. Here, we show that Shisa7, a recently identified GABAAR auxiliary subunit, modulates GABAAR activation by altering single-channel burst kinetics. These results reveal that Shisa7 primarily decreases the duration and open probability of receptor burst activity during gating, leading to accelerated GABAAR deactivation. These experiments are the first to assess the gating properties of GABAARs in the presence of an auxiliary subunit and provides a kinetic basis for how Shisa7 modifies temporal attributes of GABAergic transmission at the single-channel level.

Glucose-Stimulated Calcium Dynamics in Beta Cells From Male C57BL/6J, C57BL/6N, and NMRI Mice: A Comparison of Activation, Activity, and Deactivation Properties in Tissue Slices.

Although mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between C57BL/6J, C57BL/6N, and NMRI mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucose concentrations. Our results underline the significance of carefully choosing and reporting the strain to enable comparison and increase reproducibility, emphasize the importance of analyzing a number of different beta cell physiological parameters characterizing the response to glucose, and provide a valuable standard for future studies on beta cell calcium dynamics in health and disease in tissue slices.

Ultrasound and Photoacoustic Imaging of Laser-Activated Phase-Change Perfluorocarbon Nanodroplets

Laser-activated perfluorocarbon nanodroplets (PFCnDs) are emerging phase-change contrast agents that showed promising potential in ultrasound and photoacoustic (US/PA) imaging. Unlike monophase gaseous microbubbles, PFCnDs shift their state from liquid to gas via optical activation and can provide high US/PA contrast on demand. Depending on the choice of perfluorocarbon core, the vaporization and condensation dynamics of the PFCnDs are controllable. Therefore, these configurable properties of activation and deactivation of PFCnDs are employed to enable various imaging approaches, including contrast-enhanced imaging and super-resolution imaging. In addition, synchronous application of both acoustic and optical pulses showed a promising outcome vaporizing PFCnDs with lower activation thresholds. Furthermore, due to their sub-micrometer size, PFCnDs can be used for molecular imaging of extravascular tissue. PFCnDs can also be an effective therapeutic tool. As PFCnDs can carry therapeutic drugs or other particles, they can be used for drug delivery, as well as photothermal and photodynamic therapies. Blood barrier opening for neurological applications was recently demonstrated with optically-triggered PFCnDs. This paper specifically focuses on the activation and deactivation properties of laser-activated PFCnDs and associated US/PA imaging approaches, and briefly discusses their theranostic potential and future directions.

Deciphering Spectroscopic and Structural Insights into the Photophysical Behavior of 2,2'-Dipyridylamine: An Efficient Environment Sensitive Fluorescence Probe.

Excited state deactivation properties and the effects of solvent hydrogen bonding (HB) on the photophysical behavior of 2,2'-dypyridylamine (DPyA) were investigated by steady state and time-resolved fluorescence experiments, molecular docking, and density functional theory (DFT) calculations. In addition to the polarity effect, the contributions of solvent HB donation (HBD) acidity and HB acceptance (HBA) basicity to modulate the solvatochromic spectral properties were estimated from multiparametric linear regression analysis using Kamlet-Taft (KT) and Catalán formalisms. The importance of C-N bond torsion, leading to the trans → cis conversion, was manifested by substantial increase in DPyA fluorescence yield in the presence of cyclodextrin (CD) and glycerol. The unusually low fluorescence yield in aqueous medium was explained on the basis of synergistic effect of solvent hydrogen bonding combined with excited state conformational isomerization, which renders DPyA to be an excellent environment sensitive fluorescence reporter. The experimental results were verified with structural insights obtained from DFT calculations at B3LYP/6-311++G(d,p) level and construction of potential energy surface (PES) in the ground state as well as in the excited states.

Effects of modulation on sodium and potassium channel currents by extremely low frequency electromagnetic fields stimulation on hippocampal CA1 pyramidal cells

ABSTRACT To investigate the effects of extremely low-frequency electromagnetic fields (ELF-EMFs) stimulation on sodium channel currents (INa), transient outward potassium channel currents (IA) and delayed rectifier potassium channel currents (IK) on hippocampal CA1 pyramidal neurons of young Sprague–Dawley rats. CA1 pyramidal neurons of rat hippocampal slices were subjected to ELF-EMFs stimulation with different frequencies (15 and 50 Hz), intensities (0.5, 1 and 2 mT) and durations (10, 20 and 30 min). The INa, IA and IK of neurons were recorded by a whole-cell patch-clamp method. ELF-EMFs stimulation enhanced INa densities, and depressed IA and IK densities. In detail, INa was more sensitive to the variation of intensities and frequencies of ELF-EMFs, whereas IA and IK were mainly affected by the variation of the duration of ELF-EMFs. ELF-EMFs stimulation altered activation and deactivation properties of INa, IA and IK. ELF-EMFs stimulation plays a role as a regulator rather than an inducer for ion channels. It might change the transition probability of ion channel opening or closing, and might also change the structure and function of the ion channel which need to be proved by the further technical method.

Hysteresis effects and roughness suppression efficacy of polyethylenimine additive in Cu electrodeposition in ethaline

Deep eutectic solvents (DES) are environmentally-friendly electrolytes that are gaining interest for electrodeposition and energy storage applications. In these applications, metal electrodeposits with smooth, non-dendritic morphology are desired and thus effective strategies for suppressing roughness evolution are critically needed. A commonly employed and rather effective strategy for suppressing roughness evolution in metal electrodeposition is the use of electrolyte additives; however, the availability of such additives in DES electrolytes is limited and so is the understanding of the mechanisms through which additives suppress roughness amplification in DES media. In the present contribution, we demonstrate that polyethylenimine (PEI) is an effective electrolyte additive that suppresses roughness evolution during Cu electrodeposition in ethaline DES. PEI, due to its adsorption–deactivation properties, exhibits a unique hysteresis response during voltammetric studies of Cu electrodeposition – this response is analyzed using a mathematical model incorporating the relevant PEI transport, surface adsorption and deactivation processes. The model provides guidelines for selection of optimal conditions (e.g., PEI concentration) for effective suppression of roughness amplification in Cu electrodeposition.

Reactive Monolayers in Directed Additive Manufacturing - Area Selective Atomic Layer Deposition

Additive manufacturing generally describes a process of producing macro scale 3D-objects by the addition (or formation) of one patterned layer, of the overall object, onto a subsequent layer. In this fashion the target structure is produced in a layer-by-layer process. Selective area atomic layer deposition (SA-ALD) is, fundamentally, an additive manufacturing process where the layer-by-layer process occurs at the atomic level. A patterned surface is generated by exploiting disparate surface chemistries where a metal oxide is deposited in a targeted area. One method of enabling this process is through the use of organic monolayers that can form from a collection of well organized small molecules onto one surface and not another. These self-assembled monolayers (SAMs) can act as effective barriers to block the deposition of up to hundreds of thermal ALD cycles or can be used in combination with repair strategies to extend selective deposition capabilities. Recent efforts have focused on increasing the durability of these monolayer blocking layers. To this end we have designed SAM components that contain an aligning hydrogen bonding component that aid in directing the formation of well aligned monolayers as well as acting as a supramolecular cross-link. Furthermore, the components contain a photoactive diyne moiety that can be subsequently polymerized after irradiation to produce a robust ALD barrier. On structured surfaces this photoactive SAM achieved better deactivation properties than commercially available materials providing access to materials that produce a robust barrier in shorter formation times and withstand a great number of ALD cycles.

Selective preparation of monocyclic aromatic hydrocarbons from catalytic cracking of biomass fast pyrolysis vapors over Mo2N/HZSM-5 catalyst

Fast pyrolysis of biomass followed by catalytic cracking of pyrolysis vapors over zeolite catalysts offers a promising way to produce monocyclic aromatic hydrocarbons (MAHs). However, many polycyclic aromatic hydrocarbons (PAHs) will be formed accompanied with MAHs. To solve this problem, a series of noble metal-like catalysts prepared by Mo2N, W2N, MoP and WP supported on Hβ, HY and HZSM-5 were used for catalytic conversion of biomass fast pyrolysis vapors to prepare aromatic hydrocarbons rich in MAHs but with low PAHs. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) experiments were conducted to analyze the pyrolytic product distribution and evaluate the catalyst performance. Major aromatic hydrocarbons were quantitatively determined. Moreover, deactivation properties of catalysts were examined via recycling experiments. The results indicated that among the noble metal-like catalysts, Mo2N/HZSM-5 showed the best selectivity for MAHs. Compared with HZSM-5 catalyst, the modification with Mo2N could significantly decrease the formation of PAHs, while slightly increase MAHs, and also improve the anti-deactivation property of the catalyst. Under optimal conditions of 750 °C and catalyst-to-biomass ratio of 5, the actual yields of MAHs from HZSM-5 and Mo2N/HZSM-5 catalysts were 8.08 wt% and 8.19 wt% respectively, while the yields of PAHs were 2.37 wt% and 0.88 wt%.

A common haplotype containing functional CACNA1H variants is frequently coinherited with increased TPSAB1 copy number.

PurposeCaV3.2 signaling contributes to nociception, pruritus, gastrointestinal motility, anxiety, and blood pressure homeostasis. This calcium channel, encoded by CACNA1H, overlaps the human tryptase locus, wherein increased TPSAB1 copy number causes hereditary α-tryptasemia. Germ-line CACNA1H variants may contribute to the variable expressivity observed with this genetic trait.MethodsTryptase-encoding sequences at TPSAB1 and TPSB2, and TPSG1 and CACNA1H variants were genotyped in 46 families with hereditary α-tryptasemia syndrome. Electrophysiology was performed on tsA201 HEK cells transfected with wild-type or variant CACNA1H constructs. Effects on clinical phenotypes were interrogated in families with TPSAB1 duplications and in volunteers from the ClinSeq cohort.ResultsThree nonsynonymous variants in CACNA1H (rs3751664, rs58124832, and rs72552056) cosegregated with TPSAB1 duplications in 32/46 families and were confirmed to be in linkage disequilibrium (LD). In vitro, variant CaV3.2 had functional effects: reducing current densities, and altering inactivation and deactivation properties. No clinical differences were observed in association with the CACNA1H haplotype.ConclusionA previously unrecognized haplotype containing three functional CACNA1H variants is relatively common among Caucasians, and is frequently coinherited on the same allele as additional TPSAB1 copies. The variant CACNA1H haplotype, which in vitro imparts partial gain of function, does not result in detectable phenotypic differences in the heterozygous state.

Microbial deactivation properties of generation 2 and 4 poly(amidoamine) dendrimers on common bacteria found in the aqueous environment

The antibacterial properties of dendrimers, make them a good candidate, as a future alternative disinfectant, for water and wastewater treatment with minimal side effects. Therefore, this study was performed for evaluation of antibacterial effect of generation-2 and 4 poly(amidoamine) dendrimer (PAMAM) on the indicator bacteria found in the water resources.

EAG Domains Regulate LQT Mutant hERG Channels in Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The generation of human induced pluripotent stem (hiPS) cells provides a novel path for a wide range of disease research, including LQTS. The human ether-a-go-go related gene (hERG) encodes the α-subunit of a voltage-gated potassium channel underlying IKr. hERG potassium channels contain nearly 300 different disease-causing mutations, which can lead to long QT syndrome (LQTS). Its N-terminal region contains an eag domain, which is important for modulating channel deactivation properties. R56Q is a point mutation located in the PAS domain that is associated with LQTS, a defect known to increase the rate of deactivation profoundly. Previous work showed that R56Q profoundly increased the rate of deactivation and lessend steady-state inactivation in Xenopus oocytes and mammalian HEK293 cells. We also showed that a soluble eag domain could restore the aberrant deactivation kinetics and inactivation properties of hERG R56Q. Here, we tested whether the delivery of adenoviral eag domains could rescue hERG R56Q channels expressed in cardiomyocytes derived from human iPSCs (hiPSC-CMs). We used whole-cell patch-clamp recordings to measure currents from iPSC-CMs expressing hERG, hERG R56Q and hERG R56Q+eag domains. We found that the adenoviral delivery of hERG R56Q to iPSC-CM resulted in channels with a faster rate of deactivation compared to that of WT hERG, indicating that hERG R56Q retained its aberrant gating in cardiomyocytes. We found that adenoviral delivery of eag domains restored slow deactivation kinetics to hERG R56Q channels in iPSC-CMs. The results indicated that adenoviral-delivery of a small polypeptide (i.e. eag domain) could restore the current deficiencies in a hERG LQT-mutant channel. This result indicates that eag domain polypeptides may be a biological therapeutic for LQTS.

LYSOPHOSPHATIDIC ACID PROLONGS ACTION POTENTIAL DURATION AND INCREASES ELECTROPHYSIOLOGICAL INSTABILITY OF ADULT RABBIT VENTRICULAR MYOCARDIUM BY AUGMENTING L-TYPE CALCIUM CURRENT

ObjectivesLysophosphatidic acid (LPA) has various actions on cardiovascular system and is widely reported to modulate multiple ion currents in non-myocardiocytes, but little is known about its electrophysiological effects on cardiac...

Lysophosphatidic Acid Increases the Electrophysiological Instability of Adult Rabbit Ventricular Myocardium by Augmenting L-Type Calcium Current

Lysophosphatidic acid (LPA) has diverse actions on the cardiovascular system and is widely reported to modulate multiple ion currents in some cell types. However, little is known about its electrophysiological effects on cardiac myocytes. This study investigated whether LPA has electrophysiological effects on isolated rabbit myocardial preparations. The results indicate that LPA prolongs action potential duration at 90% repolarization (APD90) in a concentration- and frequency-dependent manner in isolated rabbit ventricular myocytes. The application of extracellular LPA significantly increases the coefficient of APD90 variability. LPA increased L-type calcium current (ICa,L) density without altering its activation or deactivation properties. In contrast, LPA has no effect on two other ventricular repolarizing currents, the transient outward potassium current (Ito) and the delayed rectifier potassium current (IK). In arterially perfused rabbit left ventricular wedge preparations, the monophasic action potential duration, QT interval, and Tpeak-end are prolonged by LPA. LPA treatment also significantly increases the incidence of ventricular tachycardia induced by S1S2 stimulation. Notably, the effects of LPA on action potentials and ICa,L are PTX-sensitive, suggesting LPA action requires a Gi-type G protein. In conclusion, LPA prolongs APD and increases electrophysiological instability in isolated rabbit myocardial preparations by increasing ICa,L in a Gi protein-dependent manner.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.

Altered HCN4 Channel C-Linker Interaction is Associated with Familial Tachycardia-Bradycardia Syndrome and Atrial Fibrillation

HCN channels underlie the pacemaker current If, involved in generation, regulation, and stabilization of sinus rhythm. HCN4 represents the dominant isotype in the sinoatrial node and channel dysfunction is associated with inherited sinus node bradycardia. Here, we report a previously undescribed HCN4 gene mutation that replaced the positively charged lysine 530 with an asparagine (HCN4-K530N) in a highly conserved region of the C-linker. Six members of a German family carrying the HCN4-K530N mutation developed tachycardia-bradycardia syndrome and persistent atrial fibrillation in an age-dependent fashion. HEK293 cell recordings using whole-cell patch clamp electrophysiology revealed that homomeric HCN4-K530N mutant and wild type channels activate at similar potentials and respond equally upon binding of cAMP. Heteromeric channels, in contrast, showed a significant hyperpolarizing shift in the half-maximal activation voltage. Moreover, the effect of cAMP on channel activation and deactivation properties was significantly increased in heteromeric channels. A comparison of mutant and wild type C-linker domain models suggests that altered subunit interactions between the A’ and B’ helices and the C’ and D’ helices of the neighboring subunit may change the intersubunit structural dynamics in heterotetramers of HCN4 wild type and mutant subunits, enforcing inhibition of channel activity by the nucleotide free cyclic nucleotide binding domain in the heterozygous situation. Thus, altered interaction of side chains is tolerated in homomeric mutant channels but interferes with wild type subunits in the heteromeric complex leading to f-channel dysfunction that promotes the development of tachycardia-bradycardia syndrome and persistent atrial fibrillation.

Molecular Determinants of Interactions between the N-Terminal Domain and the Transmembrane Core That Modulate hERG K+ Channel Gating

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4–S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4–S5 linker.

Different Molecular Phenotypes of LQT2-Linked hERG1a Mutations in the Same Amino Acid

The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunits that underlie the cardiac rapidly activating delayed rectifier K+ current (IKr). Patients with mutations of hERG1a channels are associated with type 2 long QT syndrome (LQT2), and more than 500 mutations, predominantly missense mutations, have been identified in hERG1a channels. In vitro, hERG1a mutations cause reduced hERG current (IhERG) by several mechanisms, including nonsense-mediated mRNA decay, channel protein trafficking-deficiency, abnormal channel gating and abnormal ion permeation. Two independent LQT2 clinical cases have reported hERG nucleotide mutations for tyrosine at amino acid 475 (ΔY475 and Y475C), yet the mechanisms for altered IhERG are not known. In this study, we heterologously expressed WT and these LQT2 mutations in HEK 293 cells and characterized their biochemical and biophysical properties, and additionally we studied two engineered mutations Y475F and Y475A that substitute structurally different amino acids. Western blot analysis of WT, ΔY475, Y475F and Y475A showed 135 and 155kDa protein bands, whereas Y475C showed only the 135kDa protein band, suggesting that Y475C is trafficking-deficient. Whole-cell patch clamp experiments showed that the V1/2 of the activation relation for ΔY475 shifted negatively to −29.8 ± 1.4 mV compared to WT, Y475F and Y475A (−12.4 ± 2.3, −9.3 ± 0.4 and −14.6 ± 0.4 mV, respectively). At −50 mV, Y475A and ΔY475 hERG deactivated faster compared to WT, whereas Y475F has little effect on channel deactivation kinetics. The data suggest that, 1) multiple mutations in a single amino acid exert strikingly different molecular phenotypes, 2) Y475C results in reduced IhERG due to a protein trafficking-deficient mechanism, 3) ΔY475 results in reduced IhERG due to an abnormal channel gating mechanism, and 4) the aromatic side chain of Y475 appears to modulate hERG1a channels deactivation properties.

Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome

Long-QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram and a high risk of sudden cardiac death due to ventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in the KCNQ1 gene, which encodes the repolarizing potassium channel mediating the delayed rectifier I(Ks) current. We screened a family affected by long-QT syndrome type 1 and identified an autosomal dominant missense mutation (R190Q) in the KCNQ1 gene. We obtained dermal fibroblasts from two family members and two healthy controls and infected them with retroviral vectors encoding the human transcription factors OCT3/4, SOX2, KLF4, and c-MYC to generate pluripotent stem cells. With the use of a specific protocol, these cells were then directed to differentiate into cardiac myocytes. Induced pluripotent stem cells maintained the disease genotype of long-QT syndrome type 1 and generated functional myocytes. Individual cells showed a “ventricular,” “atrial,” or “nodal” phenotype, as evidenced by the expression of cell-type–specific markers and as seen in recordings of the action potentials in single cells. The duration of the action potential was markedly prolonged in “ventricular” and “atrial” cells derived from patients with long-QT syndrome type 1, as compared with cells from control subjects. Further characterization of the role of the R190Q–KCNQ1 mutation in the pathogenesis of long-QT syndrome type 1 revealed a dominant negative trafficking defect associated with a 70 to 80% reduction in I(Ks) current and altered channel activation and deactivation properties. Moreover, we showed that myocytes derived from patients with long-QT syndrome type 1 had an increased susceptibility to catecholamine-induced tachyarrhythmia and that beta-blockade attenuated this phenotype. We generated patient-specific pluripotent stem cells from members of a family affected by long-QT syndrome type 1 and induced them to differentiate into functional cardiac myocytes. The patient-derived cells recapitulated the electrophysiological features of the disorder. (Funded by the European Research Council and others.)