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.     

shRNAs Targeting a Common KCNQ1 Variant Could Alleviate Long-QT1 Disease Severity by Inhibiting a Mutant Allele

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1. Patients heterozygous for such a mutation co-assemble both mutant and wild-type KCNQ1-encoded subunits into tetrameric Kv7.1 potassium channels. Here, we investigated whether allele-specific inhibition of mutant KCNQ1 by targeting a common variant can shift the balance towards increased incorporation of the wild-type allele to alleviate the disease in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). We identified the single nucleotide polymorphisms (SNP) rs1057128 (G/A) in KCNQ1, with a heterozygosity of 27% in the European population. Next, we determined allele-specificity of short-hairpin RNAs (shRNAs) targeting either allele of this SNP in hiPSC-CMs that carry an LQT1 mutation. Our shRNAs downregulated 60% of the A allele and 40% of the G allele without affecting the non-targeted allele. Suppression of the mutant KCNQ1 allele by 60% decreased the occurrence of arrhythmic events in hiPSC-CMs measured by a voltage-sensitive reporter, while suppression of the wild-type allele increased the occurrence of arrhythmic events. Furthermore, computer simulations based on another LQT1 mutation revealed that 60% suppression of the mutant KCNQ1 allele shortens the prolonged action potential in an adult cardiomyocyte model. We conclude that allele-specific inhibition of a mutant KCNQ1 allele by targeting a common variant may alleviate the disease. This novel approach avoids the need to design shRNAs to target every single mutation and opens up the exciting possibility of treating multiple LQT1-causing mutations with only two shRNAs.     

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.     

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).     

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.     

Pacemaker Activity of the Human Sinoatrial Node: An Update on the Effects of Mutations in HCN4 on the Hyperpolarization-Activated Current

Since 2003, several loss-of-function mutations in the HCN4 gene, which encodes the HCN4 protein, have been associated with sinus node dysfunction. In human sinoatrial node (SAN), HCN4 is the most abundant of the four isoforms of the HCN family. Tetramers of HCN subunits constitute the ion channels that conduct the hyperpolarization-activated “funny” current (I_f), which plays an important modulating role in SAN pacemaker activity. Voltage-clamp experiments on HCN4 channels expressed in COS-7, CHO and HEK-293 cells, as well as in Xenopus oocytes have revealed changes in the expression and kinetics of mutant channels, but the extent to which especially the kinetic changes would affect I_f flowing during a human SAN action potential often remains unresolved. In our contribution to the Topical Collection on Human Single Nucleotide Polymorphisms and Disease Diagnostics, we provide an updated review of the mutation-induced changes in the expression and kinetics of HCN4 channels and provide an overview of their effects on I_f during the time course of a human SAN action potential, as assessed in simulated action potential clamp experiments. Future research may solve apparent inconsistencies between data from clinical studies and data from in vitro and in silico experiments.     

Inhibition of the Akt/PKB Kinase Increases Nav1.6-Mediated Currents and Neuronal Excitability in CA1 Hippocampal Pyramidal Neurons

In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Na_v channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Na_v1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (I_Na) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Na_v1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Na_v1.6-dependent manner.     

Zingerone Modulates Neuronal Voltage-Gated Na+ and L-Type Ca2+ Currents

Zingerone (ZO), a nontoxic methoxyphenol, has been demonstrated to exert various important biological effects. However, its action on varying types of ionic currents and how they concert in neuronal cells remain incompletely understood. With the aid of patch clamp technology, we investigated the effects of ZO on the amplitude, gating, and hysteresis of plasmalemmal ionic currents from both pituitary tumor (GH₃) cells and hippocampal (mHippoE-14) neurons. The exposure of the GH₃ cells to ZO differentially diminished the peak and late components of the I_Na. Using a double ramp pulse, the amplitude of the I_Na(P) was measured, and the appearance of a hysteresis loop was observed. Moreover, ZO reversed the tefluthrin-mediated augmentation of the hysteretic strength of the I_Na(P) and led to a reduction in the I_Ca,L. As a double ramp pulse was applied, two types of voltage-dependent hysteresis loops were identified in the I_Ca,L, and the replacement with BaCl₂-attenuated hysteresis of the I_Ca,L enhanced the I_Ca,L amplitude along with the current amplitude (i.e., the I_Ba). The hysteretic magnitude of the I_Ca,L activated by the double pulse was attenuated by ZO. The peak and late I_Na in the hippocampal mHippoE-14 neurons was also differentially inhibited by ZO. In addition to acting on the production of reactive oxygen species, ZO produced effects on multiple ionic currents demonstrated herein that, considered together, may significantly impact the functional activities of neuronal 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).     

Oxidation of iodide ions by means of cyclic voltammetry

From chronovoltammetric and cyclovoltammetric polarization curves three mechanisms of iodide ions have been established: 3I^? = I^?₃ + 2e,2I^?₃ = 3I₂ + 2e and 2I^? = I₂ + 2e under certain conditions of scan rate, concentration of iodide ions and area of electrode in the aqueous solutions KI (background solution 1 N Na₂SO₄) with half-wave potentials of 0.428, 0.489 and 0.507 V by silver/silver chloride electrode.     

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare^®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH₃ cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K⁺ current (I_K(M)) with effective EC₅₀ value of 0.34 μM. The activation time constant of I_K(M) was concurrently shortened in the SOL presence, hence yielding the K_D value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of I_K(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of I_K(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated I_K(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in I_K(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K⁺ (K_M) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH₃ cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the I_K(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with K_M channels and hence in stimulating I_K(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH₃ or mHippoE-14 cells.     

Physiological Roles of the Rapidly Activated Delayed Rectifier K+ Current in Adult Mouse Heart Primary Pacemaker Activity

Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na⁺, K⁺ and Ca²⁺ that produce SAN action potentials (AP) and ‘pacemaker depolarizations’ in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca²⁺-regulated mechanism based on a requirement for phasic release of Ca²⁺ from intracellular stores and activation of an inward current-mediated by Na⁺/Ca²⁺ exchange; (ii) time- and voltage-dependent activation of Na⁺ or Ca²⁺ currents, as well as a cyclic nucleotide-activated current, I_f; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K⁺ current, often denoted I_Kr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K⁺ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K⁺ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K⁺ current can contribute to its role in generating spontaneous myogenic activity.     

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD₉₀. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (I_to) and late sodium current (I_Na,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of I_to. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly I_Na,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.     

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca²⁺ imaging, and computational modeling, we evaluated the effects of S1P on the Ca²⁺-activated K⁺ currents (I_K(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BK_Ca channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca²⁺ concentration (Ca_i) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BK_Ca was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed I_K(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of I_K(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced I_K(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BK_Ca conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited I_K(Ca) and the permissive role of S1P on the BK_Ca activity was also effectively observed in the PC12 cell system. The S1P-mediated I_K(Ca) stimulation may result from the elevated Ca_i, whereas the inhibition of BK_Ca activity by S1P appears to be direct. By the differentiated tailoring BK_Ca channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.     

Carbonato- and methanediolato(-2)-bridged nickel(II) coordination clusters from the use of N-salicylidene-4-methyl-o-aminophenol

The use of N-salicylidene-4-methyl-o-aminophenol in nickel(II) pivalate chemistry has provided access to structurally and magnetically interesting {Ni^II₆Na^I₄} and {Ni^II₆} clusters possessing carbonato and methanediolato(-2) ligands, respectively, with novel coordination modes.     

Effectiveness in Block by Dexmedetomidine of Hyperpolarization-Activated Cation Current,Independent of Its Agonistic Effect on α2-Adrenergic Receptors

Dexmedetomidine (DEX), a highly selective agonist of α₂-adrenergic receptors, has been tailored for sedation without risk of respiratory depression. Our hypothesis is that DEX produces any direct perturbations on ionic currents (e.g., hyperpolarization-activated cation current, I_h). In this study, addition of DEX to pituitary GH₃ cells caused a time- and concentration-dependent reduction in the amplitude of I_h with an IC₅₀ value of 1.21 μM and a K_D value of 1.97 μM. A hyperpolarizing shift in the activation curve of I_h by 10 mV was observed in the presence of DEX. The voltage-dependent hysteresis of I_h elicited by long-lasting triangular ramp pulse was also dose-dependently reduced during its presence. In continued presence of DEX (1 μM), further addition of OXAL (10 μM) or replacement with high K⁺ could reverse DEX-mediated inhibition of I_h, while subsequent addition of yohimbine (10 μM) did not attenuate the inhibitory effect on I_h amplitude. The addition of 3 μM DEX mildly suppressed the amplitude of erg-mediated K⁺ current. Under current-clamp potential recordings, the exposure to DEX could diminish the firing frequency of spontaneous action potentials. In pheochromocytoma PC12 cells, DEX was effective at suppressing I_h together with a slowing in activation time course of the current. Taken together, findings from this study strongly suggest that during cell exposure to DEX used at clinically relevant concentrations, the DEX-mediated block of I_h appears to be direct and would particularly be one of the ionic mechanisms underlying reduced membrane excitability in the in vivo endocrine or neuroendocrine cells.     

A Sequence Variation in GmBADH2 Enhances Soybean Aroma and Is a Functional Marker for Improving Soybean Flavor

The vegetable soybean (Glycine max L. Merr.) plant is commonly consumed in Southeast Asian countries because of its nutritional value and desirable taste. A “pandan-like” aroma is an important value-added quality trait that is rarely found in commercial vegetable soybean varieties. In this study, three novel aromatic soybean cultivars with a fragrant volatile compound were isolated. We confirmed that the aroma of these cultivars is due to the potent volatile compound 2-acetyl-1-pyrroline (2AP) that was previously identified in soybean. A sequence comparison of GmBADH1/2 (encoding an aminoaldehyde dehydrogenase) between aromatic and non-aromatic soybean varieties revealed a mutation with 10 SNPs and an 11-nucleotide deletion in exon 1 of GmBADH2 in Quxian No. 1 and Xiangdou. Additionally, a 2-bp deletion was detected in exon 10 of GmBADH2 in ZK1754. The mutations resulted in a frame shift and the introduction of premature stop codons. Moreover, genetic analyses indicated that the aromatic trait in these three varieties was inherited according to a single recessive gene model. These results suggested that a mutated GmBADH2 may be responsible for the aroma of these three aromatic soybean cultivars. The expression and function of GmBADH2 in aromatic soybean seeds were confirmed by qRT-PCR and CRISPR/Cas9. A functional marker developed on the basis of the mutated GmBADH2 sequence in Quxian No. 1 and Xiangdou was validated in an F₂ population. A perfect association between the marker genotypes and aroma phenotypes implied that GmBADH2 is a major aroma-conferring gene. The results of this study are potentially useful for an in-depth analysis of the molecular basis of 2-AP formation in soybean and the marker-assisted breeding of aromatic vegetable soybean cultivars.     

Cathodic reduction of oxygen on copper and brass

In this investigation, the electrochemical reduction of oxygen on copper and brass has been studied using the ring-disc electrode technique in solutions containing chloride, sulphate and nitrate anions and ammonium cation. The E—I curves obtained in the forward and reverse direction of polarization for copper and brass in NaCl, Na₂SO₄ and NH₄Cl solutions are similar in nature and show two waves. However in (NH₄)₂SO₄ and NH₄Cl solutions are similar in nature and show two waves. The rate of oxygen reduction is the highest in ammonium sulphate for both copper and brass whereas it is the lowest in NH₄Cl in the case of copper. In Na₂SO₄ and NaCl solutions, the rate of oxygen reduction is higher on copper than on brass. Apparent Tafel slopes for oxygen reduction obtained for copper and brass vary from 65 mV to 240 mV depending upon the medium.The two steps observed with copper disc electrode have been identified as due to the reduction of oxygen to hydrogen peroxide and reduction of H₂O₂ to OH^? ion or water depending upon the pH of the solution. In acid chloride and sulphate media no H₂O₂ was detected, which suggests direct reduction to H₂O. The diagnostic plots of I_d/I_rυs ω^{?$\text{1}\text{2}$} employed by Bockris et al indicate that in Na₂SO₄ the reduction of oxygen to H₂O₂ takes place in a parallel reaction whereas in (NH₄)₂SO₄ both direct reduction of O₂ to water (or OH^? ion) and the reduction through intermediate H₂O₂ occur.