Delayed Ventricular Repolarization and Sodium Channel Current Modification in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a severe developmental disorder that is strongly linked to mutations in the MECP2 gene. RTT has been associated with sudden unexplained death and ECG QT interval prolongation. There are mixed reports regarding QT prolongation in mouse models of RTT, with some evidence that loss of Mecp2 function enhances cardiac late Na current, I_Na,Late. The present study was undertaken in order to investigate both ECG and ventricular AP characteristics in the Mecp2^Null/Y male murine RTT model and to interrogate both fast I_Na and I_Na,Late in myocytes from the model. ECG recordings from 8–10-week-old Mecp2^Null/Y male mice revealed prolongation of the QT and rate corrected QT (QTc) intervals and QRS widening compared to wild-type (WT) controls. Action potentials (APs) from Mecp2^Null/Y myocytes exhibited longer APD₇₅ and APD₉₀ values, increased triangulation and instability. I_Na,Late was also significantly larger in Mecp2^Null/Y than WT myocytes and was insensitive to the Nav1.8 inhibitor A-803467. Selective recordings of fast I_Na revealed a decrease in peak current amplitude without significant voltage shifts in activation or inactivation V_0.5. Fast I_Na ‘window current’ was reduced in RTT myocytes; small but significant alterations of inactivation and reactivation time-courses were detected. Effects of two I_Na,Late inhibitors, ranolazine and GS-6615 (eleclazine), were investigated. Treatment with 30 µM ranolazine produced similar levels of inhibition of I_Na,Late in WT and Mecp2^Null/Y myocytes, but produced ventricular AP prolongation not abbreviation. In contrast, 10 µM GS-6615 both inhibited I_Na,Late and shortened ventricular AP duration. The observed changes in I_Na and I_Na,Late can account for the corresponding ECG changes in this RTT model. GS-6615 merits further investigation as a potential treatment for QT prolongation in RTT.     

Functional Characterization of Two Novel Mutations in SCN5A Associated with Brugada Syndrome Identified in Italian Patients

Background. Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac disease characterized by “coved type” ST-segment elevation in the right precordial leads, high susceptibility to ventricular arrhythmia and a family history of sudden cardiac death. The SCN5A gene, encoding for the cardiac voltage-gated sodium channel Nav1.5, accounts for ~20–30% of BrS cases and is considered clinically relevant. Methods. Here, we describe the clinical findings of two Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. Results. Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu mutation significantly reduced peak sodium current density without affecting channel membrane localization. Changes in the gating properties of expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 wild-type channel. Conclusions. Overall, our results suggest that the degree of loss-of-function shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.     

Effectiveness of Columbianadin,a Bioactive Coumarin Derivative,in Perturbing Transient and Persistent INa

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca²⁺ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH₃ cells and HL-1 atrial cardiomyocytes). GH₃-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na⁺ current (I_Na) with effective IC₅₀ of 14.7 or 2.8 µM, respectively. The inactivation time course of I_Na activated by short depolarization became fastened in the presence of CBN with estimated K_D value of 3.15 µM. The peak I_Na diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of I_Na. CBN (10 µM) shifted the midpoint of inactivation curve of I_Na to the leftward direction. The CBN-mediated inhibition of peak I_Na exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent I_Na enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K⁺ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak I_Na and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in I_Na. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies I_Na in electrically excitable cells.     

Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of I_Ca showed no difference in the magnitude of current. Measurements of I_Na reveal a 60% reduction in I_Na density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of I_Na was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of I_Na due to a mutation in PKP2 coupled with and a gain of function in I_K,ATP due to a mutation in ABCC9.     

Characterization in Inhibitory Effectiveness of Carbamazepine in Voltage-Gated Na+ and Erg-Mediated K+ Currents in a Mouse Neural Crest-Derived (Neuro-2a) Cell Line

Carbamazepine (CBZ, Tegretol^®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na⁺ current (I_Na) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., I_Na and erg-mediated K⁺ current [I_K(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na in a concentration-dependent manner with effective IC₅₀ of 56 and 18 μM, respectively. The overall current–voltage relationship of I_Na(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of I_Na (I_Na(W)) evoked by the short ascending ramp pulse (V_ramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate I_Na, augmented the strength of the voltage-dependent hysteresis (Hys_(V)) of persistent I_Na (I_Na(P)) in response to the isosceles-triangular V_ramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of I_Na(P)’s Hys_(V). With a two-step voltage protocol, the recovery of I_Na(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of I_Na(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K⁺ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na⁺ (Na_V) channel predicted the ability of CBZ to bind to some amino-acid residues in Na_V due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the I_Na (I_Na(T), I_Na(L), I_Na(W), and I_Na(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on I_Na(L) is larger than that on I_Na(T). Collectively, the magnitude and gating of Na_V channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.     

The Evidence for Effective Inhibition of INa Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of CaV Channels

Mirogabalin (MGB, Tarlige^®), an inhibitor of the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH₃) cells, we found that MGB was effective at suppressing the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of the voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner, with an effective IC₅₀ of 19.5 and 7.3 μM, respectively, while the K_D value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of I_Na(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of I_Na(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (I_Na(W)) or resurgent I_Na (I_Na(R)) evoked by the respective ascending or descending ramp pulse (V_ramp) was reduced during cell exposure to MGB. MGB-induced attenuation in I_Na(W) or I_Na(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate I_Na. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent I_Na in response to the isosceles triangular V_ramp. The cumulative inhibition of I_Na(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of Ca_V channels, the Na_V channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.     

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease

Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na⁺ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.     

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca²⁺-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na⁺ currents in myocytes. Hearts from Trpm4^−/− mice presented increased sensitivity towards mexiletine, a Na⁺ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na⁺ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na⁺ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Na_v1.5 function in murine cardiac myocytes.     

Inhibitory Effectiveness of Gomisin A,a Dibenzocyclooctadiene Lignan Isolated from Schizandra chinensis,on the Amplitude and Gating of Voltage-Gated Na+ Current

Gomisin A (Gom A), a lignan isolated from Schisandra chinensis, has been reported produce numerous biological activities. However, its action on the ionic mechanisms remains largely unanswered. The present experiments were undertaken to investigate the possible perturbations of Gom A or other related compounds on different types of membrane ionic currents in electrically excitable cells (i.e., pituitary GH₃ and pancreatic INS-1 cells). The exposure to Gom A led to the differential inhibition of peak and end-pulse components of voltage-gated Na⁺ current (I_Na) in GH₃ cells with effective IC₅₀ of 6.2 and 0.73 μM, respectively. The steady-state inactivation curve of I_Na in the presence of Gom A was shifted towards a more hyperpolarized potential. However, neither changes in the overall current-voltage relationship nor those for the gating charge of the current were demonstrated. The application of neither morin (10 μM) nor hesperidin (10 μM) perturbed the strength of I_Na, while sesamine could suppress it. However, in the continued presence of Gom A, the addition of sesamine failed to suppress I_Na further. Gom A also effectively suppressed the strength of persistent I_Na activated by long ramp voltage command, and further application of tefluthrin effectively attenuated Gom A-mediated inhibition of the current. The presence of Gom A mildly inhibited erg-mediated K⁺ current, while a lack of change in the amplitude of hyperpolarization-activated cation current was observed in its presence. Under cell-attached current recordings, the exposure to Gom A resulted in the decreased firing of spontaneous action currents with a minimal change in AC amplitude. In pancreatic INS-1 cells, the presence of Gom A was also noticed to inhibit peak and end-pulse components of I_Na differentially with the IC₅₀ of 5.9 and 0.84 μM, respectively. Taken together, the emerging results presented herein provide the evidence that Gom A can differentially inhibit peak and sustained I_Na in endocrine cells (e.g., GH₃ and INS-1 cells).     

Characterization in Effective Stimulation on the Magnitude,Gating, Frequency Dependence,and Hysteresis of INa Exerted by Picaridin (or Icaridin), a Known Insect Repellent

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.     

Evidence for Inhibitory Perturbations on the Amplitude,Gating, and Hysteresis of A-Type Potassium Current,Produced by Lacosamide,a Functionalized Amino Acid with Anticonvulsant Properties

Lacosamide (Vimpat^®, LCS) is widely known as a functionalized amino acid with promising anti-convulsant properties; however, adverse events during its use have gradually appeared. Despite its inhibitory effect on voltage-gated Na⁺ current (I_Na), the modifications on varying types of ionic currents caused by this drug remain largely unexplored. In pituitary tumor (GH₃) cells, we found that the presence of LCS concentration-dependently decreased the amplitude of A-type K⁺ current (I_K(A)) elicited in response to membrane depolarization. The I_K(A) amplitude in these cells was sensitive to attenuation by the application of 4-aminopyridine, 4-aminopyridine-3-methanol, or capsaicin but not by that of tetraethylammonium chloride. The effective IC₅₀ value required for its reduction in peak or sustained I_K(A) was calculated to be 102 or 42 µM, respectively, while the value of the dissociation constant (K_D) estimated from the slow component in I_K(A) inactivation at varying LCS concentrations was 52 µM. By use of two-step voltage protocol, the presence of this drug resulted in a rightward shift in the steady-state inactivation curve of I_K(A) as well as in a slowing in the recovery time course of the current block; however, no change in the gating charge of the inactivation curve was detected in its presence. Moreover, the LCS addition led to an attenuation in the degree of voltage-dependent hysteresis for I_K(A) elicitation by long-duration triangular ramp voltage commands. Likewise, the I_K(A) identified in mouse mHippoE-14 neurons was also sensitive to block by LCS, coincident with an elevation in the current inactivation rate. Collectively, apart from its canonical action on I_Na inhibition, LCS was effective at altering the amplitude, gating, and hysteresis of I_K(A) in excitable cells. The modulatory actions on I_K(A), caused by LCS, could interfere with the functional activities of electrically excitable cells (e.g., pituitary tumor cells or hippocampal neurons).     

Kinetics of the SCN/(SCN)2 couple on platinum in acetonitrile

The electrode kinetics of the SCN⁻/(SCN)₂ system has been studied on platinum in the temperature range from −12 to −3°C at different ammonium thiocyanate concentrations in acetonitrile in the presence of 0·1 M [(C₂H₅)₄N]ClO₄. Quasi-steady E/I curves with a Pt rotating disk electrode and E/I curves at different linear potential-sweep rates were recorded under different experimental conditions.     

SCN5A Variants as Genetic Arrhythmias Triggers for Familial Bileaflet Mitral Valve Prolapse

Mitral valve prolapse (MVP) is a common valvular heart defect with variable outcomes. Several studies reported MVP as an underestimated cause of life-threatening arrhythmias and sudden cardiac death (SCD), mostly in young adult women. Herein, we report a clinical and genetic investigation of a family with bileaflet MVP and a history of syncopes and resuscitated sudden cardiac death. Using family based whole exome sequencing, we identified two missense variants in the SCN5A gene. A rare variant SCN5A:p.Ala572Asp and the well-known functional SCN5A:p.His558Arg polymorphism. Both variants are shared between the mother and her daughter with a history of resuscitated SCD and syncopes, respectively. The second daughter with prodromal MVP as well as her healthy father and sister carried only the SCN5A:p.His558Arg polymorphism. Our study is highly suggestive of the contribution of SCN5A mutations as the potential genetic cause of the electric instability leading to ventricular arrhythmias in familial MVP cases with syncope and/or SCD history.     

Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin,a Pyrethroid Insecticide

Deltamethrin (DLT) is a type-II pyrethroid ester insecticide used in agricultural and domestic applications as well as in public health. However, transmembrane ionic channels perturbed by this compound remain largely unclear, although the agent is thought to alter the gating characteristics of voltage-gated Na⁺ (Na_V) channel current. In this study, we reappraised whether and how it and other related compounds can make any further modifications on voltage-gated Na⁺ current (I_Na) in pituitary tumor (GH₃) cells. Cell exposure to DLT produced a differential and dose-dependent stimulation of peak (transient, I_Na(T)) or sustained (late, I_Na(L)) I_Na; consequently, the EC₅₀ value required for DLT-stimulated I_Na(T) or I_Na(L) was determined to be 11.2 or 2.5 μM, respectively. However, neither the fast nor slow component in the inactivation time constant of I_Na(T) activated by short depolarizing pulse was changed with the DLT presence; conversely, tefluthrin (Tef), a type-I pyrethroid insecticide, can accentuate I_Na with a slowing in inactivation time course of the current. The I_Na(L) augmented by DLT was attenuated by further application of either dapagliflozin (Dapa) or amiloride, but not by chlorotoxin. During pulse train (PT) stimulation, with the Tef or DLT presence, the cumulative inhibition of I_Na(T) became slowed; moreover, following PT stimuli, a large tail current with a slowly recovering process was observed. Alternatively, during rapid depolarizing pulse, the amplitude of I_Na(L) and tail I_Na (I_Na(Tail)) for each depolarizing pulse became progressively increased by adding DLT, not by Tef. The recovery time constant following PT stimulation with continued presence of Tef or DLT was shortened by further addition of Dapa. The voltage-dependent hysteresis (Hys_(V)) of persistent I_Na was differentially augmented by Tef or DLT. Taken together, the magnitude, gating, frequency dependence, as well as Hys_(V) behavior of I_Na exerted by the presence of DLT or Tef might exert a synergistic impact on varying functional activities of excitable cells in culture or in vivo.     

Veratridine Can Bind to a Site at the Mouth of the Channel Pore at Human Cardiac Sodium Channel NaV1.5

The cardiac sodium ion channel (Na_V1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na⁺ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits Na_V1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on Na_V1.5, we perform docking calculations and high-throughput electrophysiology experiments in the present study. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of Na_V1.4 and Na_V1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at Na_V1.5.     

Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3′-Diol), a Dietary Xanthophyll Carotenoid

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, I_h [or funny current, I_f]). In the present study, GH₃-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in I_h amplitude with an IC₅₀ value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of I_h in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked I_h, respectively. The voltage-dependent anti-clockwise hysteresis of I_h responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K⁺ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of I_h would be an ionic mechanism underlying its changes in membrane excitability.     

Near-infrared quantum cutting in Ho3+, Yb3+-codoped BaGdF5 nanoparticles via first- and second-order energy transfers

Infrared quantum cutting involving Yb³⁺ 950–1,000 nm (² F_5/2 → ² F_7/2) and Ho³⁺ 1,007 nm (⁵S₂,⁵F₄ → ⁵I₆) as well as 1,180 nm (⁵I₆ → ⁵I₈) emissions is achieved in BaGdF₅: Ho³⁺, Yb³⁺ nanoparticles which are synthesized by a facile hydrothermal route. The mechanisms through first- and second-order energy transfers were analyzed by the dependence of Yb³⁺ doping concentration on the visible and infrared emissions, decay lifetime curves of the ⁵ F₅ → ⁵I₈, ⁵S₂/⁵F₄ → ⁵I₈, and ⁵ F₃ → ⁵I₈ of Ho³⁺, in which a back energy transfer from Yb³⁺ to Ho³⁺ is first proposed to interpret the spectral characteristics. A modified calculation equation for quantum efficiency of Yb³⁺-Ho³⁺ couple by exciting at 450 nm was presented according to the quantum cutting mechanism. Overall, the excellent luminescence properties of BaGdF₅: Ho³⁺, Yb³⁺ near-infrared quantum cutting nanoparticles could explore an interesting approach to maximize the performance of solar cells.