An Etiological Foxp2 Mutation Impairs Neuronal Gain in Layer VI Cortico-Thalamic Cells through Increased GABAB/GIRK Signaling.

A rare mutation affecting the Forkhead-box protein P2 (FOXP2) transcription factor causes a severe monogenic speech and language disorder. Mice carrying an identical point mutation to that observed in affected patients (Foxp2+/R552H mice) display motor deficits and impaired synaptic plasticity in the striatum. However, the consequences of the mutation on neuronal function, in particular in the cerebral cortex, remain little studied. Foxp2 is expressed in a subset of Layer VI cortical neurons. Here, we used Ntsr1-EGFP mice to identify Foxp2+ neurons in the mouse auditory cortex ex vivo. We studied the functional impact of the R552H mutation on the morphologic and functional properties of Layer VI cortical neurons from Ntsr1-EGFP; Foxp2+/R552H male and female mice. The complexity of apical, but not basal dendrites was significantly lower in Foxp2+/R552H cortico-thalamic neurons than in control Foxp2+/+ neurons. Excitatory synaptic inputs, but not inhibitory synaptic inputs, were decreased in Foxp2+/R552H mice. In response, homeostatic mechanisms would be expected to increase neuronal gain, i.e., the conversion of a synaptic input into a firing output. However, the intrinsic excitability of Foxp2+ cortical neurons was lower in Foxp2+/R552H neurons. A-type and delayed-rectifier (DR) potassium currents, two putative transcriptional targets of Foxp2, were not affected by the mutation. In contrast, GABAB/GIRK signaling, another presumed target of Foxp2, was increased in mutant neurons. Blocking GIRK channels strongly attenuated the difference in intrinsic excitability between wild-type (WT) and Foxp2+/R552H neurons. Our results reveal a novel role for Foxp2 in the control of neuronal input/output homeostasis.SIGNIFICANCE STATEMENT Mutations of the Forkhead-box protein 2 (FOXP2) gene in humans are the first known monogenic cause of a speech and language disorder. The Foxp2 mutation may directly affect neuronal development and function in neocortex, where Foxp2 is expressed. Brain imaging studies in patients with a heterozygous mutation in FOXP2 showed abnormalities in cortical language-related regions relative to the unaffected members of the same family. However, the role of Foxp2 in neocortical neurons is poorly understood. Using mice with a Foxp2 mutation equivalent to that found in patients, we studied functional modifications in auditory cortex neurons ex vivo We found that mutant neurons exhibit alterations of synaptic input and GABAB/GIRK signaling, reflecting a loss of neuronal homeostasis.

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression.

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 µM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 µM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and Ikr blocker.

New in vitro model for proarrhythmia safety screening: IKs inhibition potentiates the QTc prolonging effect of IKr inhibitors in isolated guinea pig hearts.

INTRODUCTION: Preclinical in vivo QT measurement as a proarrhythmia essay is expensive and not reliable enough. The aim of the present study was to develop a sensitive, cost-effective, Langendorff perfused guinea pig heart model for proarrhythmia safety screening. METHODS: Low concentrations of dofetilide and cisapride (inhibitors of the rapid delayed rectifier potassium current, IKr) were tested alone and co-perfused with HMR-1556 (inhibitor of the slow delayed rectifier potassium current, IKs) in Langendorff perfused guinea pig hearts. The electrocardiographic rate corrected QT (QTc) interval, the Tpeak-Tend interval and the beat-to-beat variability and instability (BVI) of the QT interval were determined in sinus rhythm. RESULTS: Dofetilide and HMR-1556 alone or co-perfused, prolonged the QTc interval by 20±2%, 10±1% and 55±10%, respectively. Similarly, cisapride and HMR-1556 alone or co-perfused, prolonged the QTc interval by 11±3%, 11±4% and 38±6%, respectively. Catecholamine-induced fast heart rate abolished the QTc prolonging effects of the IKr inhibitors, but augmented the QTc prolongation during IKs inhibition. None of the drug perfusions increased significantly the Tpeak-Tend interval and the sinus BVI of the QT interval. DISCUSSION: IKs inhibition increased the QTc prolonging effect of IKr inhibitors in a super-additive (synergistic) manner, and the QTc interval was superior to other proarrhythmia biomarkers measured in sinus rhythm in isolated guinea pig hearts. The effect of catecholamines on the QTc facilitated differentiation between IKr and IKs inhibitors. Thus, QTc measurement in Langendorff perfused guinea pig hearts with pharmacologically attenuated repolarization reserve and periodic catecholamine perfusion seems to be suitable for preclinical proarrhythmia screening.

The positive frequency-dependent electrophysiological effects of the IKur inhibitor XEN-D0103 are desirable for the treatment of atrial fibrillation.

BACKGROUND: Selective inhibitors of Kv1.5 channels are being developed for the treatment of atrial fibrillation (AF). OBJECTIVES: The purpose of this study was to investigate the effects of the highly selective Kv1.5 inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety. METHODS: Intracellular APs (stimulation rates 1-5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study. RESULTS: Depending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 µM) shortened action potential duration at 90% repolarization (APD90) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD90 and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD90 and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 µM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase ∆∆QTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms. CONCLUSION: APD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.

Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. CONCLUSIONS: Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 µg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

Crude oil impairs cardiac excitation-contraction coupling in fish.

Crude oil is known to disrupt cardiac function in fish embryos. Large oil spills, such as the Deepwater Horizon (DWH) disaster that occurred in 2010 in the Gulf of Mexico, could severely affect fish at impacted spawning sites. The physiological mechanisms underlying such potential cardiotoxic effects remain unclear. Here, we show that crude oil samples collected from the DWH spill prolonged the action potential of isolated cardiomyocytes from juvenile bluefin and yellowfin tunas, through the blocking of the delayed rectifier potassium current (I(Kr)). Crude oil exposure also decreased calcium current (I(Ca)) and calcium cycling, which disrupted excitation-contraction coupling in cardiomyocytes. Our findings demonstrate a cardiotoxic mechanism by which crude oil affects the regulation of cellular excitability, with implications for life-threatening arrhythmias in vertebrates.

Developmental changes in action potential prolongation by K+-channel blockers in chick myocardium.

The effects of K(+)-channel blockers on the action potential duration of the myocardium were examined in isolated right ventricles from the 7 - 10-day-old, 11 - 13-day-old, and 14 - 20-day-old embryo and 1 - 7-day-old hatched chicks. E-4031 significantly prolonged action potential duration at all developmental stages examined; the prolongation was largest in the 11 - 13-day-old embryo and was accompanied by early after-depolarizations. Chromanol 293B showed smaller prolongation at all stages examined. Terfenadine prolonged action potential duration in the 11 - 13-day-old embryo, but not in other stages. Thus, the chick ventricular myocardium changes its repolarization properties during development.

Dexrazoxane protects the heart from acute doxorubicin-induced QT prolongation: a key role for I(Ks).

INTRODUCTION: Doxorubicin, an anthracycline widely used in the treatment of a broad range of tumours, causes acute QT prolongation. Dexrazoxane has been shown to prevent the QT prolongation induced by another anthracycline, epirubicin, but has not yet been reported to prevent that induced by doxorubicin. Thus, the present study was designed to test whether the acute QT effects induced by doxorubicin could be blocked by dexrazoxane and to explore the mechanism. Results were compared with those obtained with a reference human ether-a-go-go (hERG) channel blocker, moxifloxacin. METHODS: The effects of moxifloxacin (100 microM) and doxorubicin (30 microM), with or without dexrazoxane (from 3 to 30 microM), have been evaluated on the QTc interval in guinea-pig isolated hearts and on I(Kr) (rapid component of the delayed rectifier current) and I(Ks) (slow component of the delayed rectifier current) currents stably expressed in human embryonic kidney 293 cells. RESULTS: Moxifloxacin (100 microM), a potent hERG blocker, prolonged QTc by 22%, and this effect was not prevented by dexrazoxane. Doxorubicin (30 microM) also prolonged QTc by 13%, did not significantly block hERG channels and specifically inhibited I(Ks) (IC(50): 4.78 microM). Dexrazoxane significantly reduced the doxorubicin-induced QTc prolongation and prevented doxorubicin-induced inhibition of I(Ks). CONCLUSION AND IMPLICATIONS: Doxorubicin acutely prolonged the QT interval in guinea-pig heart by selective I(Ks) blockade. This effect was prevented by dexrazoxane. This result is important because it illustrates the danger of neglecting I(Ks) in favour of hERG screening alone, for early preclinical testing for possible induction of torsade de pointes.

Effect of GBE50 on delayed rectifier potassium current of ventricular myocytes in ischemic guinea pig / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Ginkgo biloba extract 50 (GBE50) is a new multicomponent drug with a polyvalent action extracted from the leave of Ginkgo biloba. The aim of this experiment was to study the effects of GBE50 on delayed rectifier potassium current (I(K)) in ventricular myocytes under normal and simulated ischemia conditions in guinea pigs.</p><p><b>METHODS</b>Single ventricular myocytes were isolated by an enzymatic dissociation method. I(K) were recorded by whole-cell patch clamp technique in voltage clamp mode. GBE50 was added to the perfusion chamber from low to high concentrations (25, 50,100 mg/L) in normal condition. Different concentrations of GBE50 (25, 50, 100 mg/L) were prepared with simulated ischemic fluid.</p><p><b>RESULTS</b>(1) Under normal condition, 100 mg/L GBE50 decreased I(K) (n = 7, P < 0.05). (2) Under ischemia condition, it was observed that I(K) was inhibited (n = 8, P < 0.05). (3) Perfusion with ischemia solution containing 50 mg/L (n = 8, P > 0.05) and 100 mg/L GBE50 (n = 6, P > 0.05) could reverse the decrease of I(K).</p><p><b>CONCLUSION</b>GBE50 significantly decreased I(K) in a concentration-dependent manner. GBE50 could alleviate the electrophysiological heterogeneity of myocardium to prevent ischemic myocardium from arrhythmia.</p>

[Effect of GBE50 on delayed rectifier potassium current of ventricular myocytes in ischemic guinea pig].

OBJECTIVE: Ginkgo biloba extract 50 (GBE50) is a new multicomponent drug with a polyvalent action extracted from the leave of Ginkgo biloba. The aim of this experiment was to study the effects of GBE50 on delayed rectifier potassium current (I(K)) in ventricular myocytes under normal and simulated ischemia conditions in guinea pigs. METHODS: Single ventricular myocytes were isolated by an enzymatic dissociation method. I(K) were recorded by whole-cell patch clamp technique in voltage clamp mode. GBE50 was added to the perfusion chamber from low to high concentrations (25, 50,100 mg/L) in normal condition. Different concentrations of GBE50 (25, 50, 100 mg/L) were prepared with simulated ischemic fluid. RESULTS: (1) Under normal condition, 100 mg/L GBE50 decreased I(K) (n = 7, P < 0.05). (2) Under ischemia condition, it was observed that I(K) was inhibited (n = 8, P < 0.05). (3) Perfusion with ischemia solution containing 50 mg/L (n = 8, P > 0.05) and 100 mg/L GBE50 (n = 6, P > 0.05) could reverse the decrease of I(K). CONCLUSION: GBE50 significantly decreased I(K) in a concentration-dependent manner. GBE50 could alleviate the electrophysiological heterogeneity of myocardium to prevent ischemic myocardium from arrhythmia.

Cardiac repolarization: insights from mathematical modeling and electrocardiographic imaging (ECGI).

Cardiac repolarization is a complex rate dependent process. At the cellular level, it depends on a delicate dynamic balance of ion channel currents. At the heart level, it is spatially heterogeneous, leading to spatial gradients of potential and excitability. This article provides insights into the molecular mechanisms of the delayed rectifiers I(Kr) (rapid) and I(Ks) (slow) that underlie effective function of these channels as repolarizing currents during the cardiac action potential (AP). We also provide noninvasive images of cardiac repolarization in humans. Methodologically, computational biology is used to simulate ion channel function and to incorporate it into a model of the cardiac cell. ECG imaging (ECGI) is applied to normal subjects and Wolff-Parkinson-White (WPW) patients to obtain epicardial maps of repolarization. The simulations demonstrate that I(Kr) and I(Ks) generate their peak current late during the AP, where they effectively participate in repolarization. I(Kr) maximizes the current by fast inactivation and gradual recovery during the AP. I(Ks) does so by generating an available reserve of channels in closed states from which the channels can open rapidly. ECGI shows that in the human heart, normal repolarization epicardial potential maps are static with 40 ms dispersion between RV and LV. In WPW, ECGI located the accessory pathway(s) and showed a large base-to-apex repolarization gradient that resolved to normal one month post-ablation, demonstrating presence of "cardiac memory". We conclude that computational biology can provide a mechanistic link across scales, from the molecular functioning of ion channels to the cellular AP. ECGI can noninvasively image human cardiac repolarization and its alteration by disease and interventions. This property makes it a potential tool for noninvasive risk stratification and evaluation of treatment by drugs and devices.

Quantitative analysis of T-wave morphology increases confidence in drug-induced cardiac repolarization abnormalities: evidence from the investigational IKr inhibitor Lu 35-138.

This study investigates repolarization changes induced by a new candidate drug to determine whether a composite electrocardiographic (ECG) measure of T-wave morphology could be used as a reliable marker to support the evidence of abnormal repolarization, which is indicated by QT interval prolongation. Seventy-nine healthy subjects were included in this parallel study. After a baseline day during which no drug was given, 40 subjects received an I(Kr)-blocking antipsychotic compound (Lu 35-138) on 7 consecutive days while 39 subjects received placebo. Resting ECGs were recorded and used to determine a combined measure of repolarization morphology (morphology combination score [MCS]), based on asymmetry, flatness, and notching. Replicate measurements were used to determine reliable change and study power for both measures. Lu 35-138 increased the QTc interval with corresponding changes in T-wave morphology as determined by MCS. For subjects taking Lu 35-138, T-wave morphology was a more reliable indicator of I(Kr) inhibition than QTcF (chi(2) = 20.3, P = .001). At 80% study power for identifying a 5-millisecond placebo-adjusted change from baseline for QTcF, the corresponding study power for MCS was 93%. As a covariate to the assessment of QT interval liability, MCS offered important additive information to the effect of Lu 35-138 on cardiac repolarization.

Inhibitory effects of isoliquiritigenin and licorice extract on voltage-dependent K(+) currents in H9c2 cells.

The effect of isoliquiritigenin (ISL), a component of licorice, on the voltage-dependent, ultra-rapidly activating delayed-rectifier K(+) current (IKur) was examined in H9c2 cells, a cell-line derived from rat cardiac myoblasts. IKur was recorded using the whole-cell patch clamp method with a pipette solution containing 140 mM K(+). Depolarizing voltage pulses of 200-ms duration were given with 10-mV steps every 10 s from -40 mV holding potential. ISL inhibited IKur in a concentration-dependent manner. The median inhibitory concentration (IC(50)) of ISL was approximately 0.11 microM and the Hill coefficient was 0.71. Using CHO cells expressing Kv1.5 IKur channels, ISL also inhibited Kv1.5 IKur, but less potently than the IKur current in H9c2 cells. Furthermore, in H9c2 cells, the licorice extract itself inhibited IKur in a manner similar to ISL. We conclude that ISL, one component of licorice, is a potent inhibitor of K(+) channels, which specifically in H9c2 cells could be Kv2.1, and that this inhibition may be involved in various pharmacological effects of licorice.

Discovery of talatisamine as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons.

Blocking specific K+ channels has been proposed as a promising strategy for the treatment of neurodegenerative diseases. Using a computational virtual screening approach and electrophysiological testing, we found four Aconitum alkaloids are potent blockers of the delayed rectifier K+ channel in rat hippocampal neurons. In the present study, we first tested the action of the four alkaloids on the voltage-gated K+, Na+ and Ca2+ currents in rat hippocampal neurons, and then identified that talatisamine is a specific blocker for the delayed rectifier K+ channel. External application of talatisamine reversibly inhibited the delayed rectifier K+ current (IK) with an IC50 value of 146.0+/-5.8 microM in a voltage-dependent manner, but exhibited very slight blocking effect on the voltage-gated Na+ and Ca2+ currents even at the high concentration of 1-3 mM. Moreover, talatisamine exerted a significant hyperpolarizing shift of the steady-state activation, but did not influence the steady state inactivation of IK and its recovery from inactivation, suggesting that talatisamine had no allosteric action on IK channel and was a pure blocker binding to the external pore entry of the channel. Our present study made the first discovery of potent and specific IK channel blocker from Aconitum alkaloids. It has been argued that suppressing K+ efflux by blocking IK channel may be favorable for Alzheimer's disease therapy. Talatisamine can therefore be considered as a leading compound worthy of further investigations.

The hERG potassium channel and hERG screening for drug-induced torsades de pointes.

Drug-induced torsades de pointes (TdP) arrhythmia is a major safety concern in the process of drug design and development. The incidence of TdP tends to be low, so early pre-clinical screens rely on surrogate markers of TdP to highlight potential problems with new drugs. hERG (human ether-à-go-go-related gene, alternative nomenclature KCNH2) is responsible for channels mediating the 'rapid' delayed rectifier K+ current (IKr) which plays an important role in ventricular repolarization. Pharmacological inhibition of native IKr and of recombinant hERG channels is a shared feature of diverse drugs associated with TdP. In vitro hERG assays therefore form a key element of an integrated assessment of TdP liability, with patch-clamp electrophysiology offering a 'gold standard'. However, whilst clearly necessary, hERG assays cannot be assumed automatically to provide sufficient information, when considered in isolation, to differentiate 'safe' from 'dangerous' drugs. Other relevant factors include therapeutic plasma concentration, drug metabolism and active metabolites, severity of target condition and drug effects on other cardiac ion channels that may mitigate or exacerbate effects of hERG blockade. Increased understanding of the nature of drug-hERG channel interactions may ultimately help eliminate potential hERG blockade early in the design and development process. Currently, for promising drug candidates integration of data from hERG assays with information from other pre-clinical safety screens remains essential.

An automated electrophysiology serum shift assay for K(V) channels.

The presence of serum in biological samples often negatively impacts the quality of in vitro assays. However, assays tolerant of serum are useful for assessing the in vivo availability of a small molecule for its target. Electrophysiology assays of ion channels are notoriously sensitive to serum because of their reliance on the interaction of the plasma membrane with a recording electrode. Here we investigate the tolerance of an automated electrophysiology assay for a voltage-gated potassium (K(V)) channel to serum and purified plasma proteins. The delayed rectifier channel, K(V)2.1, stably expressed in Chinese hamster ovary cells produces large, stable currents on the IonWorks Quattro platform (MDS Analytical Technologies, Sunnyvale, CA), making it an ideal test case. K(V)2.1 currents recorded on this platform are highly resistant to serum, allowing recordings in as high as 33% serum. Using a set of compounds related to the K(V) channel blocker, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone, we show that shifts in compound potency with whole serum or isolated serum proteins can be reliably measured with this assay. Importantly, this assay is also relatively insensitive to plasma, allowing the creation of a bioassay for inhibitors of K(V)2.1 channel activity. Here we show that such a bioassay can quantify the levels of the gating modifier, guangxitoxin-1E, in plasma samples from mice dosed with the peptide. This study demonstrates the utility of using an automated electrophysiology platform for measuring serum shifts and for bioassays of ion channel modulators.

Basal and angiotensin II-inhibited neuronal delayed-rectifier K+ current are regulated by thioredoxin.

In previous studies, we determined that macrophage migration inhibitory factor (MIF), acting intracellularly via its intrinsic thiol-protein oxidoreductase (TPOR) activity, stimulates basal neuronal delayed-rectifier K(+) current (I(Kv)) and inhibits basal and angiotensin (ANG) II-induced increases in neuronal activity. These findings are the basis for our hypothesis that MIF is a negative regulator of ANG II actions in neurons. MIF has recently been recategorized as a member of the thioredoxin (Trx) superfamily of small proteins. In the present study we have examined whether Trx influences basal and ANG II-modulated I(Kv) in an effort to determine whether the Trx superfamily can exert a general regulatory influence over neuronal activity and the actions of ANG II. Intracellular application of Trx (0.8-80 nM) into rat hypothalamic/brain stem neurons in culture increased neuronal I(Kv), as measured by voltage-clamp recordings. This effect of Trx was abolished in the presence of the TPOR inhibitor PMX 464 (800 nM). Furthermore, the mutant protein recombinant human C32S/C35S-Trx, which lacks TPOR activity, failed to alter neuronal I(Kv). Trx applied at a concentration (0.08 nM) that does not alter basal I(Kv) abolished the inhibition of neuronal I(Kv) produced by ANG II (100 nM). Given our observation that ANG II increases Trx levels in neuronal cultures, it is possible that Trx (like MIF) has a negative regulatory role over basal and ANG II-stimulated neuronal activity via modulation of I(Kv). Moreover, these data suggest that TPOR may be a general mechanism for negatively regulating neuronal activity.

Comparison of four rate-correction algorithms for the ventricular repolarization period in assessing net effects of IKr blockers in dogs.

The utility of corrected and uncorrected QT interval changes for assessing net repolarization delay by I(Kr) (a rapid component of delayed rectifier K(+) currents) blockers was assessed in halothane-anesthetized dogs using the electrocardiogram and monophasic action potential (MAP) recordings with electrical ventricular pacing. Intravenous administration of dl-sotalol (0.2 - 2 mg/kg) prolonged the MAP duration and RR interval, while terfenadine (3 mg/kg) increased the MAP duration but transiently shortened RR interval. The order of correlation coefficient between the MAP duration at a pacing cycle length of 400 ms and MAP duration itself or that with arithmetical correction was uncorrected > Van de Water = Matsunaga > Fridericia > Bazett. These results suggest that Matsunaga's and Van de Water's formulae would better predict the net repolarization delay in the in vivo canine model. Also, the risk of drug candidates that may prolong the QT interval should be judged by change in uncorrected QT interval as well as corrected QT interval.

Effects of Liuwei Dihuang decoction on ion channels and synaptic transmission in cultured hippocampal neuron of rat.

The effect of Liuwei Dihuang decoction (LW), a traditional Chinese medicine (TCM) prescription, on voltage-dependent currents and synaptic transmission were investigated in cultured hippocampal neurons of rat by whole-cell patch clamp recording technique. After application with serum from LW-treated rats, termed LW-containing serum (LWCS) for 48 h, the amplitude of delay rectifying K+ current (IK) and voltage-gated Ca2+ current (ICa) decreased. While the frequency of spontaneous excitatory post-synaptic current (sEPSC) and miniature excitatory post-synaptic current (mEPSC) increased significantly. Yet the amplitude of voltage-depended Na+ current (INa) and transient outward K+ current (IA), membrane capacitance and resistance remained unchanged. The results indicated that LWCS possessed the effect of modulating or improving neuronal and synaptic function, which possibly contribute to the cognition enhancing effect of LW.