Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Modulation of IKs channel-PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization.

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (IKs ) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the IKs channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1-specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to IKs reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced IKs and concurrently decreased the arginine methylation of KCNQ1, a pore-forming α-subunit. Evidence presented here indicates that furamidine decreased IKs mainly by lowering the affinity of IKs channels for the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2 ), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine-induced IKs reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated IKs activity through channel-PIP2 interaction, thereby restricting excessive cardiac action potential.

Hypoxia with inflammation and reperfusion alters membrane resistance by dynamically regulating voltage-gated potassium channels in hippocampal CA1 neurons.

Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (IDR) can explain the input resistance variability. Furthermore, voltage-dependent inactivation of A-type potassium (IA) channels shifted in the depolarizing direction during Hypo-Inf and reverted to normal upon reperfusion without a significant alteration in the maximum current density. Our results indicate that changes in the input resistance, and consequently excitability, caused by Hypo-Inf and reperfusion are at least partially regulated by the availability and voltage dependence of KV channels. Moreover, these results suggest that selective KV channel modulators can be used as potential neuroprotective drugs to minimize hypoxia- and reperfusion-induced neuronal damage.

Usefulness of an IKr blocker for ablation of non-pulmonary vein ectopies that are unmappable due to easily initiated atrial fibrillation.

PURPOSE: When atrial fibrillation (AF) is initiated by a single or several non-pulmonary vein (PV) trigger ectopic beats, mapping the ectopy is often difficult, requiring a number of electrical cardioversion applications. Nifekalant is a rapidly activating delayed rectifier potassium channel (IKr) blocker which may suppress AF initiation without inhibiting ectopy development, thereby allowing the target ectopy to be mapped. To assess the efficacy of nifekalant in the ablation of non-PV ectopies that are unmappable due to easily initiated AF. METHODS: Eleven consecutive patients were administered nifekalant to map a non-PV ectopy that was unmappable using a conventional method due to easily initiated AF. Nifekalant was intravenously administered as a bolus dose of 0.2 mg/kg, and electrical cardioversion was delivered. Additional boluses of 0.2 mg/kg were repeatedly administered until AF initiation was prevented or until the appearance of significant prolongation of QT interval. RESULTS: AF suppression without inhibition of ectopy development was achieved in 7 patients. These patients had a higher rate of acute elimination of the ectopy than the remaining 4 patients without AF suppression (7 [100%] vs. 1 [25%] patients, p = 0.024). In addition, patients with suppression of AF initiation had a higher AF recurrence-free rate than those without (7 [100%] vs. 1 [25%] patients, p = 0.024). CONCLUSION: Nifekalant administration appears useful in the ablation of non-PV ectopies that easily initiate AF.

Modulation of Ion Channels in the Superior Cervical Ganglion Neurons by Myocardial Ischemia and Fluvastatin Treatment.

Background: The superior cervical ganglion (SCG) of the autonomic nervous system plays an important role in different cardiovascular diseases. In this study, we investigated the effects of ischemia and fluvastatin treatment on the ion channel characteristics of SCG neurons in a rabbit myocardial ischemia (MI) model. Methods: MI was induced by abdominal subcutaneous injections of isoproterenol (ISO). The properties of the delayed rectifier potassium channel current (IK ), sodium channel current (INa ), and action potential (APs) on isolated SCG neurons in the control, MI-7d, MI-14d, fluvastatin-7d (fluvastatin pretreated 14 days and treated 7 days after ISO-induced MI), and fluvastatin-14d (fluvastatin pretreated 14 days and treated 14 days after ISO-induced MI) groups were studied. In addition, the RNA expressions of KCNQ3 and SCN9A in the SCG tissue were determined by performing real-time PCR. Intracellular calcium concentration was monitored using laser scanning confocal microscopy. Results: Compared with the control group, the current amplitude of IK and INa were increased in the MI-7d and MI-14d groups. KCNQ3 RNA (corresponding to channel proteins of IK ) expression and SCN9A RNA (corresponding to channel proteins of INa ) expression were also increased in MI groups. Activation and inactivation curves for INa in the two MI groups shifted negatively compared with the control group. These changes were reversed by fluvastatin treatment. Intracellular calcium concentration in SCG neurons was not altered significantly by MI or fluvastatin treatment. By contrast, increased AP amplitude and shortened APD90 were observed in the MI-7d and MI-14d groups. These changes were reversed in the fluvastatin-treated MI group. Conclusion: Fluvastatin treatment partly reversed the characteristics of SCG neurons in MI. The ion channel of SCG neurons could be one of the potential targets of fluvastatin in treating coronary heart diseases.

Effect of down-regulation of IKs repolarization-reserve on ventricular arrhythmogenesis in a guinea pig model of cardiac hypertrophy / 中南大学学报(医学版)

Objective:To observe the changes of rapidly activated delayed rectifier potassium channel (IKs) and slowly activated delayed rectifier potassium channel (IKs) in cardiac hypertrophy and to evaluate the effects of IKs and IKs blocker on the incidence ofventricular arrhythmias in guinea pigs with left ventricular hypertrophy (LVH).Methods:Guinea pigs were divided into a sham operation group and a left ventricular hypertrophy (LVH) group.LVH model was prepared.Whole cell patch-clamp technique was used to record IKr and IKs tail currents in a guinea pig model with LVH.The changes of QTc and the incidence rate of ventricular arrhythmias in LVH guinea pigs were observed by using the IKr and IKs blockers.Results:Compared with cardiac cells in the control group,the interventricular septal thickness at end systole (IVSs),left ventricular posterior wall thickness at end systole (LVPWs),QTc interval and cell capacitance in guinea pigs with LVH were significantly increased (P＜0.05);while IKs densities were significantly reduced [+60 mV:(0.36±0.03) pA/pF vs (0.58±0.05) pA/pF,P＜0.01].However,LVH exerted no significant effect on IKr densities.IKr blocker markedly prolonged the QTc interval (P＜0.01) and increased the incidence of ventricular arrhythmias in guinea pigs with LVH compared with the control guinea pigs.In contrast,IKs blocker produced modest increase in QTc interval in guinea pigs of control group with no increase in LVH animals.IKs blocker did not induce ventricular arrhythmias incidence in either control or LVH animals.Conclusion:The cardiac hypertrophy-induced arrhythmogenesis is due to the down-regulation of IKs.

Effects of Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum on chemotactic migration of alveolar macrophages and inters regions macrophages in rats.

OBJECTIVE: To observe the effects of serum containing Mahuang (Herba Ephedra Sinica) or Wuweizi (Fructus Schisandrae Chinensis) on the migration of alveolar macrophages (AM) and interstitial macrophages (IM) from normal rats, and to analyze and compare the mechanisms leading to cell migration differences. METHODS: Rats were randomly divided into three groups: Mahuang (Herba Ephedra Sinica), Wuweizi (Fructus Schisandrae Chinensis), and blank serum. After treatment with the herbs, serum was extracted from the rats. AM and IM were isolated from normal rats and cultured. The effects of Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum on normal rat AM and IM chemotactic migration were determined by transwell assays. The CC chemokine receptor (CCR) 2, CCR5, voltage-gated Kvl. 3 K+ channel (Kv1. 3), and voltage-gated Kvl. 5 K+ channel (Kv1. 5) protein levels were analyzed by western blotting. RESULTS: The migration quantities of AM and IM in the Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) medicated serum groups were significantly higher than those in the blank serum group (P < 0.01). Compared with the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group, the migration quantity of cultured rat AM in the Mahuang (Herba Ephedra Sinica) medicated serum group was significantly increased (P < 0.01). Meanwhile, compared with the Mahuang (Herba Ephedra Sinica) medicated serum group, the migration quantity of cultured rat IM in the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group was significantly increased (P < 0.01). CCR2, CCR5, Kv1. 3, and Kv1. 5 proteins were expressed on the AM cell surface, and showed significantly higher expression in the Mahuang (Herba Ephedra Sinica) medicated serum group compared with the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group. In contrast, CCR5, Kv1.3, and Kv1.5 proteins were expressed on the IM cell surface, and showed significantly higher expression in the Wuweizi (Fructus Schisandrae Chinensis) medicated serum group compared with the Mahuang (Herba Ephedra Sinica) medicated serum group. CONCLUSION: Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) can promote AM and IM migration ability, with Mahuang (Herba Ephedra Sinica) targeting AM more apparently and Wuweizi (Fructus Schisandrae Chinensis) targeting IM more apparently. The mechanism may be that, by stimulating cells, Mahuang (Herba Ephedra Sinica) and Wuweizi (Fructus Schisandrae Chinensis) promote expression of CCR2 and CCR5 receptors on the AM and IM cell surface, which pass signals to Kvl.3 and Kvl.5 ion channels, leading to changes in the cytoskeleton, and ultimately promoting chemotactic cell migration.

Effects of allocryptopine on outward potassium current and slow delayed rectifier potassium current in rabbit myocardium.

OBJECTIVE: Allocryptopine (ALL) is an effective alkaloid of Corydalis decumbens (Thunb.) Pers. Papaveraceae and has proved to be anti-arrhythmic. The purpose of our study is to investigate the effects of ALL on transmural repolarizing ionic ingredients of outward potassium current (I to) and slow delayed rectifier potassium current (I Ks). METHODS: The monophasic action potential (MAP) technique was used to record the MAP duration of the epicardium (Epi), myocardium (M) and endocardium (Endo) of the rabbit heart and the whole cell patch clamp was used to record I to and I Ks in cardiomyocytes of Epi, M and Endo layers that were isolated from rabbit ventricles. RESULTS: The effects of ALL on MAP of Epi, M and Endo layers were disequilibrium. ALL could effectively reduce the transmural dispersion of repolarization (TDR) in rabbit transmural ventricular wall. ALL decreased the current densities of I to and I Ks in a voltage and concentration dependent way and narrowed the repolarizing differences among three layers. The analysis of gating kinetics showed ALL accelerated the channel activation of I to in M layers and partly inhibit the channel openings of I to in Epi, M and Endo cells. On the other hand, ALL mainly slowed channel deactivation of I Ks channel in Epi and Endo layers without affecting its activation. CONCLUSIONS: Our study gives partially explanation about the mechanisms of transmural inhibition of I to and I Ks channels by ALL in rabbit myocardium. These findings provide novel perspective regarding the anti-arrhythmogenesis application of ALL in clinical settings.

Voltage-gated potassium channels in cognitive dysfunction / 医学研究生学报

The growing number of cognitive dysfunction patients is bringing heavy mental and financial burdens to the society and families.Voltage-gated potassium channels (Kv), which consist of delayed rectifier potassium channels and transient outward po -tassium channels , are involved in the incidence of cognitive dysfunction .This review summarized the role of Kv channels in cognitive dysfunction and their relationship with N-methyl-D-aspartic acid receptors ( NMDARs) that play an important role in the process of learning and memory .

Kv2 channel regulation of action potential repolarization and firing patterns in superior cervical ganglion neurons and hippocampal CA1 pyramidal neurons.

Kv2 family "delayed-rectifier" potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60-80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from -70 mV, but not -80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at -70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation.

Sequence Alterations of I(Ks) Potassium Channel Genes in Kazakhstani Patients with Atrial Fibrillation.

INTRODUCTION: Atrial fibrillation (AF) is the most common sustained arrhythmia, and it results in significant morbidity and mortality. However, the pathogenesis of AF remains unclear to date. Recently, more pieces of evidence indicated that AF is a multifactorial disease resulting from the interaction between environmental factors and genetics. Recent studies suggest that genetic mutation of the slow delayed rectifier potassium channel (I(Ks)) may underlie AF. OBJECTIVE: To investigate sequence alterations of I(Ks) potassium channel genes KCNQ1, KCNE1 and KCNE2 in Kazakhstani patients with atrial fibrillation. METHODS: Genomic DNA of 69 cases with atrial fibrillation and 27 relatives were analyzed for mutations in all protein-coding exons and their flanking splice site regions of the genes KCNQ1 (NM_000218.2 and NM_181798.1), KCNE1 (NM_000219.2), and KCNE2 (NM_172201.1) using bidirectional sequencing on the ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). RESULTS: In total, a disease-causing mutation was identified in 39 of the 69 (56.5%) index cases. Of these, altered sequence variants in the KCNQ1 gene accounted for 14.5% of the mutations, whereas a KCNE1 mutation accounted for 43.5% of the mutations and KCNE2 mutation accounted for 1.4% of the mutations. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. We found two sequence variants in KCNQ1 exon 13 (S546S G1638A) and exon 16 (Y662Y, C1986T) in ten patients (14.5%). In KCNE1 gene in exon 3 mutation, S59G A280G was observed in 30 of 69 patients (43.5%) and KCNE2 exon 2 T10K C29A in 1 patient (1.4%). Genetic cascade screening of 27 relatives to the 69 index cases with an identified mutation revealed 26.9% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. CONCLUSION: In this cohort of Kazakhstani index cases with AF, a disease-causing mutation was identified in 56.5 % of the referred patients. Further screening of mutations in other genes encoding cardiac ion channels is needed to clarify possible disease causing and founder mutations in Kazakhstani atrial fibrillation patients.

The Effects of Protein Kinase C (PKC) on the Tension of Normal and Passively Sensitized Human Airway Smooth Muscle and the Activity of Voltage-dependent Delayed Rectifier Potassium Channel (Kv) / 华中科技大学学报（医学）（英德文版）

The effects of protein kinase C (PKC) on the tension and the activity of voltage-dependent delayed rectifier potassium channel (Kv) were examined in normal and passively sensitized human airway smooth muscle (HASM), by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential (Em) were also detected. The results showed that phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group (P＜0.05), and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO (a PMA menstruum)-treated group (P＜0.01). This effect could be blocked by Ro31-8220 (P＜0.01). It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

The Effects of Botulinum Toxin-A on Calcium Channel and KDR Channel of the Detrusor Muscle in Rat Bladder Body / 대한비뇨기과학회지

PURPOSE: Botulium toxin-A (BoTx A) is useful in treating detrusor-sphincter dyssynergia, detrusor hyperreflexia, and refractory overactive bladder. Only the blocking action of acetycholine (ACh) release from nerve endings is the well known aspect of the action mechanism. The aim of this study is to investigate the effects of BoTx A on the detrusor muscle itself. MATERIALS AND METHODS: Sprague-Dawley rats were divided into 3 groups: the control group, the low dose injection group (1unit/ml of BoTx A, 0.5cc), and the high dose injection group (5units/ml of BoTx A, 0.5cc). All rats were either injected with normal saline (control group) or BoTx A (injection groups). Ten days after injection, a strip of the detrusor muscle was harvested. Contraction and relaxation responses of the strips were measured by an isometric force transducer. Contractions were induced by various concentrations of ACh, bethanechol, phenylephrine (PE), high concentrations of potassium (35, 70, 105, 140mM), tetraethylammonium (TEA, 0.1, 1, 10mM), 4-aminopyridine (4-AP, a delayed rectifier K+ antagonist, 0.1, 1, 10mM), and Bay K8644 (a L-type voltage dependent calcium channel opener, 0.1, 1, 10mM). The results were analyzed by ANOVA and the Student's t test. RESULTS: Contractions of the strips were noted when concentrations were above 1mM for TEA and above 0.1mM for 4-AP. A high dose injection as well as a low dose injection of BoTx A had no significant effects on the Ach or bethanechol-induced contractions of the strips compared to the control group. Denervation supersensitivity was not found in the injection groups after the Ach and bethanechol treatments, but the contractility was decreased in high concentrations of potassium (70, 105, 100mM), TEA (10mM), 4-AP (10mM), and Bay K8644 in both the high and low dose injection groups. There was no significant difference in the decrease of contractility between the high and low dose groups with the exception of the Bay K8644 1M treatment. CONCLUSIONS: BoTx seems to have some direct effects on decreasing the contractility of the detrusor muscle by increasing the delayed rectifier K+ channel activity and decreasing the L-type voltage dependent calcium channel activity.

Effect of morphine on the Kv channel in bronchial smooth muscle in guinea-pigs / 中国药理学通报

Aim To explore the effect of the morphine(MP) on the Kv channel in bronchial smooth muscle in guinea-pigs (GPBSM) and expressions of the Kv 1.5 mRNA and protein . Methods The effect of the MP on the Kv channel was studied by patch clamp recording in GPBSM and expressions of the Kv 1.5 mRNA and protein were measured by Western-blot and RT-PCR techniques. Results Morphine remarkably restrained the Kv channel current and the expression of the Kv 1.5 mRNA and protein in cultured GPBSM (n=6, each group, P

Effects of berberine on potassium current of guinea pig colonic smooth muscle cells / 中国药理学通报

AIM To clarify the mechanism of berberine on mobility diarrhea by investigating the effects of berberine on calcium-activated potassium current I K(Ca) and delayed-rectifier potassium channel current I K(V) of guinea pig colonic smooth muscle cells. METHODS Single guinea pig colonic smooth muscle cells was isolated by collagenase; The effects of berberine on I K(Ca) and I K(V) were detected by using patch clamp technique, under the conventional whole cell patch clamp mode. RESULTS 10, 50, 100 ?mol?L -1 berberine inhibited I K(Ca) of guinea pig colonic smooth muscle cells significantly (P