Calcium Channel Currents Research Articles

Bifurcation Analysis on a Generation of Early Afterdepolarization in a Mathematical Cardiac Model

Electrical activity occurs in the cell membrane of cardiomyocytes. This electrical activity forms the action potential that generates pumping of the heart. An abnormality in the action potential turns into arrhythmia, which may cause sudden death. Studies of arrhythmias using mathematical models are important to reduce the risk of sudden death. In this study, we investigate bifurcations related to the generation of early afterdepolarizations (EADs) in a mathematical model. We clarify the transition process from a normal state to a persistent EAD through a transient EAD while changing only one parameter (multiple of conductance of L-type calcium channel current) value. The dependence of the transient EAD generation on parameters is shown through bifurcation analysis in a [Na]i-parameterized system.

Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism.

BackgroundVentricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α2-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated.MethodsA conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (If), potassium (K+) channel currents (IK1 and IKr), sodium (Na+) channel currents (INa), and calcium (Ca2+) channel currents (ICa) in ventricular-like hiPSC-CMs.ResultsDMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC50) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD90). DMED also inhibited the amplitudes of the INa and ICa without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na+ and Ca2+ channel activation at potential –40 to –20 mv, and –20 mv. DMED increased the time constant of inactivation of the Na+ and Ca2+ channels. However, DMED did not affect the IK1, IKr, If, and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na+ and Ca2+ channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine.ConclusionsDMED could inhibit the frequency of spontaneous action potentials and decrease the INa and ICa of hiPSC-CMs via mechanisms that were independent of the α2-adrenoceptor, the imidazoline receptor, and the α1-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases’ membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.

Effect of Evodiamine on Rat Colonic Hypermotility Induced by Water Avoidance Stress and the Underlying Mechanism.

Background and AimEVO is a natural alkaloid that reportedly has potential value in regulating gastrointestinal motility, but this conclusion remains controversial, and the molecular mechanism is unclear. In this study, we aimed to explore the effect of short-chain fatty acids on rat colonic hypermotility induced by water avoidance stress and the underlying mechanism.MethodsWe constructed a hypermotile rat model by chronic water avoidance stress, and Western blot was used to detect the protein level of nNOS in colon tissue. The organ bath and multichannel physiological signal acquisition systems were used to examine the spontaneous contractions of smooth muscle strips. The whole‐cell patch‐clamp technique was used to investigate L‐type voltage‐dependent calcium and BKCa channel currents in colonic smooth muscle cells.ResultsEVO inhibited the spontaneous contractions of colonic smooth muscle strips in a dose-dependent manner. Moreover, EVO decreased the fecal output induced by chronic water avoidance stress. TTX did not block the inhibitory effect of EVO on spontaneous colon contractions, while L-NNA, a selective nNOS synthase inhibitor, did partially abolish this inhibitory effect. The protein expression of nNOS in the colon tissues of rats administered EVO was significantly increased compared to that in control rats. EVO reversibly inhibited the L-type calcium channel current without changing the steady-state activation or inactivation in colonic smooth muscle cells. EVO significantly inhibited the BKCa current but did not change the shape of the I‐V curves.ConclusionEVO inhibits gastrointestinal motility by inhibiting L-type calcium and BKCa channels in colonic smooth muscle cells and indirectly interacting with nNOS.

The Role of S-Acylation in the Regulation of Store-Operated Calcium Entry

Store-operated calcium entry (SOCE) is critical for T cell-mediated immunity, specifically, for T cell activation, clonal expansion, and differentiation. In T cells, SOCE is facilitated by the plasma membrane (PM) localized protein Orai1 and the endoplasmic reticulum (ER) membrane localized protein STIM1. Upon T cell receptor activation, ER calcium stores are depleted, leading to STIM1 activation, which undergoes a conformational change, allowing it to interact with Orai1 at ER/PM junctions. This interaction enables Orai1 gating, leading to calcium entry into the cell. Although the importance of Orai1 and STIM1 in SOCE has been established, it is still unclear what mechanisms regulate the formation of Orai1/STIM1 complexes at ER/PM junctions. We have found that, among several other regulatory T cell proteins, both Orai1 and STIM1 are S-acylated. S-acylation is the post-translational lipidation of a cysteine residue via a labile thioester bond. Using biochemical, electrophysiological, and advanced imaging approaches, we have found that S-acylation of Orai1 and STIM1 is critical for their function. Using acyl-biotin exchange, we determined not only are Orai1 and STIM1 S-acylated, but also the S-acylation of Orai1 is rapid, transient, and calcium-dependent. To further study the importance of S-acylation on SOCE, we used whole cell recording to show that the S-acylation of both Orai1 and STIM1 is important for proper calcium channel currents. Lastly, after performing total internal reflection fluorescence, Förster resonance energy transfer, and Fura-2 calcium imaging, we determined acylation-deficient Orai1 significantly decreases SOCE and does not efficiently interact with STIM1 to form functional calcium channels. This work describes a previously unknown signaling pathway that regulates SOCE in T cells, and contributes to T cell-mediated immunity.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents.

BackgroundConotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. MethodsVr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells.Results About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents.ConclusionsVr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.

Dandelion extract relaxes mouse airway smooth muscle by blocking VDLCC and NSCC channels

BackgroundAsthma is one of the main intractable diseases recognized by the international medical community. The current widely used bronchodilators for asthma—β2-adrenal receptor agonists—have limited therapeutic effects, necessitating the development of novel antiasthma drugs with increased efficacy and fewer adverse effects. In this study, we investigated the relaxant effects and underlying mechanism of an ethyl acetate extract from dandelion (EAED) on mouse airway smooth muscle.MethodsThe effects of EAED on agonist-induced precontraction in mouse airway smooth muscle were evaluated with force measurement. Mouse lung slices were used to study the effects of EAED on bronchial smooth muscle. The intracellular Ca2+ concentration was measured using a calcium imaging system. L-type voltage-dependent calcium channel (VDLCC) and non-selective cationic channel (NSCC) currents were measured by patch-clamp. The lung functions of healthy and asthmatic mouse groups were assessed via the forced oscillation technique.ResultsEAED inhibits acetylcholine-induced sustained contractions of whole airway smooth muscle by inhibiting VDLCCs, NSCCs, and some unknown channels, reduces the agonist-induced increase in the cytosolic free Ca2+ concentration in airway smooth muscle cells, blocks VDLCC and NSCC currents, and relieves the respiratory resistance of healthy and asthmatic mice.ConclusionsEAED may have potential beneficial effects on mitigating asthma attacks.

AMPK mediates regulation of voltage-gated calcium channels by leptin in isolated neurons from arcuate nucleus.

Neuronal control of the energy homeostasis requires the arcuate nucleus of the hypothalamus. This structure integrates peripheral and central signals concerning the energy state of the body. It comprises two populations of neurons releasing anorexigenic and orexigenic peptides, among others. Both populations are regulated by leptin, an anorexigenic hormone, released by white adipose tissue. Voltage-gated calcium entry is critical to promote neurotransmitter and hormone release. It is already known that calcium channel current is inhibited by leptin in orexigenic neurons. However, fine-tuning details of calcium channel regulation in arcuate nucleus by leptin remain to be elucidated. This work aimed to investigate whether 5' adenosine monophosphate-activated protein kinase (AMPK) underlies the leptin-induced inhibition of calcium channels. By using patch-clamping methods, immunocytochemical, and biochemical reagents, we recorded calcium channel currents in orexigenic neuropeptide Y neurons of the arcuate nucleus of rats. Consistently, leptin inhibition of the calcium channel current was not only prevented by AMPK inhibition with Compound C but also hampered with 5-aminoimidazole-4-carboxamide ribonucleoside. Furthermore, leptin selectively inhibited L-type calcium channel current amplitude without major changes in voltage dependence or current kinetics. These results support for the first time the key role of AMPK in the maintenance and regulation of voltage-gated calcium channels. Together, they advance our understanding of the regulation of calcium channels in the central nervous system and emerging questions concerning food intake and energy balance.NEW & NOTEWORTHY Our results readily support the hypothesis that AMPK is responsible for the maintenance of the calcium current and mediates the fine-tuning modulation of the leptin response. The novelty of these results strengthens the critical role of AMPK in the general energy balance and homeostasis.

Ionic Mechanisms of Disopyramide Prolonging Action Potential Duration in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1.

Short QT syndrome (SQTS) is associated with tachyarrhythmias and sudden cardiac death. So far, only quinidine has been demonstrated to be effective in patients with SQTS type 1(SQTS1). The aim of this study was to investigate the mechanisms of disopyramide underlying its antiarrhythmic effects in SQTS1 with the N588K mutation in HERG channel. Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1 and a healthy donor, patch clamp, and calcium imaging measurements were employed to assess the drug effects. Disopyramide prolonged the action potential duration (APD) in hiPSC-CMs from a SQTS1-patient (SQTS1-hiPSC-CMs). In spontaneously beating SQTS1-hiPSC-CMs challenged by carbachol plus epinephrine, disopyramide reduced the arrhythmic events. Disopyramide enhanced the inward L-type calcium channel current (ICa-L), the late sodium channel current (late INa) and the Na/Ca exchanger current (INCX), but it reduced the outward small-conductance calcium-activated potassium channel current (ISK), leading to APD-prolongation. Disopyramide displayed no effects on the rapidly and slowly activating delayed rectifier and ATP-sensitive potassium channel currents. In hiPSC-CMs from the healthy donor, disopyramide reduced peak INa, ICa-L, IKr, and ISK but enhanced late INa and INCX. The results demonstrated that disopyramide may be effective for preventing tachyarrhythmias in SQTS1-patients carrying the N588K mutation in HERG channel by APD-prolongation via enhancing ICa-L, late INa, INCX, and reducing ISK.

Prenatal hypoxia attenuated contraction of offspring coronary artery associated with decreased PKCβ Ser660 phosphorylation and intracellular calcium

AimsPrenatal hypoxia (PH) could affect peripheral vascular tone of the offspring, thus increasing the risk of cardiovascular diseases in adult. However, it's still unknown whether functions of coronary arteries (COA) in adult offspring would be influenced by PH. The present study aimed at effects of PH on vascular tone of COA and its related mechanisms. MethodsCoronary arteries of adult offspring exposed to hypoxic or normoxic circumstances during gestational day 5 to 21 were collected. Wire myograph system, whole-cell patch clamp technique, IonOptix MyoCam system, PCR, and western blot were used to detect vascular function of adult offspring COA. Key findingsPH significantly attenuated serotonin- and phorbol 12, 13-dibutyrate (PDBu)-induced constriction. Iberiotoxin potentiated PDBu-induced constriction and the effect was augmented by PH, however, no significant differences were found in whole-cell BKCa channel currents and its protein expression. Nifedipine inhibited PDBu-mediated constriction and the inhibitory effect was reduced in PH group, and whole-cell calcium channel current was decreased in offspring COA. Besides, PH reduced the capability of calcium release from the endoplasmic reticulum in COA. The phosphorylated PKCβ protein expression at Ser660 site, not Thr641 site, was significantly decreased in PH offspring. Chronic hypoxia during pregnancy attenuated PDBu-mediated constriction in offspring COA, presumably through decreased phosphorylated PKCβ at serine660 sites and decreased intracellular calcium-related weaker PKC activation. SignificanceThe findings provided new information on the influence of prenatal hypoxia on COA, and suggested potential use of PKCβ-serine660 for early prevention of coronary heart diseases in developmental origins.

Enhanced KCl-mediated contractility and Ca2+ sensitization in porcine collateral-dependent coronary arteries persist after exercise training.

We have previously reported enhanced Ca2+ sensitivity of coronary arteries that is dependent upon collateral circulation for their blood supply. For the current study, we hypothesized that small collateral-dependent arteries would exhibit an enhanced KCl-mediated contractile response attributable to Ca2+ sensitization and increased Ca2+ channel current. Ameroid constrictors were surgically placed around the left circumflex (LCX) artery of female Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-trained (treadmill run; 5 days/wk; 14 wk) groups. Small coronary arteries (150-300 μm luminal diameter) were isolated from myocardial regions distal to the collateral-dependent LCX and the nonoccluded left anterior descending arteries. Contractile tension and simultaneous measures of both tension and intracellular free Ca2+ levels (fura-2) were measured in response to increasing concentrations of KCl. In addition, whole cell Ca2+ currents were also obtained. Chronic occlusion enhanced contractile responses to KCl and increased Ca2+ sensitization in collateral-dependent compared with nonoccluded arteries of both sedentary and exercise-trained pigs. In contrast, smooth muscle cell Ca2+ channel current was not altered by occlusion or exercise training. Ca2+/calmodulin-dependent protein kinase II (CaMKII; inhibited by KN-93, 0.3-1 μM) contributed to the enhanced contractile response in collateral-dependent arteries of sedentary pigs, whereas both CaMKII and Rho-kinase (inhibited by hydroxyfasudil, 30 μM or Y27632, 10 μM) contributed to increased contraction in exercise-trained animals. Taken together, these data suggest that chronic occlusion leads to enhanced contractile responses to KCl in collateral-dependent coronary arteries via increased Ca2+ sensitization, a response that is further augmented with exercise training.NEW & NOTEWORTHY Small coronary arteries distal to chronic occlusion displayed enhanced contractile responses, which were further augmented after exercise training and attributable to enhanced calcium sensitization without alterations in calcium channel current. The calcium sensitization mediators Rho-kinase and CaMKII significantly contributed to enhanced contraction in collateral-dependent arteries of exercise-trained, but not sedentary, pigs. Exercise-enhanced contractile responses may increase resting arterial tone, creating an enhanced coronary flow reserve that is accessible during periods of increased metabolic demand.

Effects of Secretome from Fat Tissues on Ion Currents of Cardiomyocyte Modulated by Sodium-Glucose Transporter 2 Inhibitor.

Sodium-glucose transporter 2 (SGLT2) inhibitors were shown to decrease mortality from cardiovascular diseases in the EMPA-REG trial. However, the effects of empagliflozin (EMPA) for cardiac arrhythmia are not yet clarified. A total of 20 C57BL/6J mice were divided into four groups: (1) The control group were fed standard chow, (2) the metabolic syndrome (MS) group were fed a high-fat diet, (3) the empagliflozin (EMPA) group were fed a high-fat diet and empagliflozin 10 mg/kg daily, and (4) the glibenclamide (GLI) group were fed a high-fat diet and glibenclamide 0.6 mg/kg daily. All mice were sacrificed after 16 weeks of feeding. H9c2 cells were treated with adipocytokines from the pericardial and peripheral fat from the study groups. The delayed-rectifier potassium current (IK) and L-type calcium channel current (ICa,L) were measured by the whole-cell patch clamp techniques. Adipocytokines from the peripheral and pericardial fat tissues of mice with MS could decrease the IK and increase the ICa,L of cardiomyocytes. After treating adipocytokines from pericardial fat, the IK in the EMPA and GLI groups were significantly higher than that in the MS group. The IK of the EMPA group was also significantly higher than the GLI group. The ICa,L of the EMPA and GLI groups were significantly decreased overload compared with that of the MS group. However, there was no significant difference of IK and ICa,L among study groups after treating adipocytokines from peripheral fat. Adipocytokines from pericardial fat but not peripheral fat tissues after EMPA therapy attenuated the effects of IK decreasing and ICa,L increasing in the MS cardiomyocytes, which may contribute to anti-arrhythmic mechanisms of sodium-glucose transporter 2 (SGLT2) inhibitors.

Preventing role of warmth-promotion needling in reducing intracellular Ca2＋ overload of in vitro hippocampal neuronal cells of vascular dementia rats

To investigate the underlying mechanisms of warming-promotion needling (WPN) for vascular dementia (VD) by observing its effect on the expression of L-type calcium channel current (Ica.L), changes of intracellular [Ca2＋]i in neurons and pathomorphological changes of brain in VD rats. One hundred and eight male SD rats were randomized into normal control, model, medication, WPN, uniform reinforcing-reducing needling (URRN), and needle-twirling groups (n＝18 in each group). The VD model was established by repeated occlusion and reperfusion of the bilateral common carotid arteries. Rats of the medication group were treated by gavage of Nimodipine twice a day for 14 days. For rats of the 3 acupuncture groups, WPN or URRN or needle-twirling was applied to "Dazhui" (GV14), "Baihui"(GV20) and "Shuigou"(GV26) for 30 min, once a day for 14 days. Changes of Ica.L in hippocampal neurons were observed by whole-cell patch clamp technique, and levels of [Ca2＋]i in neuronal cells of living brain slice of hippocampal CA1 area were observed by laser confocal microscope. Histopathological changes of hippocampal CA1 area were observed under light microscope after H．E. stain. Compared with the normal control group, the current density of Ica.L, and the intracellular[Ca2＋]i levels were significantly increased (P<0.05), and the I-V curves of Ica.L was apparently shifted down in the model group. In comparison with the model group, the current density of Ica.L was obviously reduced (P<0.05), the I-V curves of Ica.L shifted up and the intracellular Ca2＋ content notably decreased in the medication, WPN, URRN and needle-twirling groups (P<0.05). The effect of WPN was significantly superior to those of URRN and needle-twiiling (P<0.05). H．E. stain showed a reduction in the number of hippocampal neurons, dilation of intercellular space, swelling of cell body, karyopyknosis, disordered arrangement and increased number of gliacytes in the model group, which was apparently milder in the medication and 3 acupuncture groups (the WPN group in particular). The WPN can reduce Ica.L current density and [Ca2＋]i content of hippocampal neurons in brain slices of VD rats, which may contribute to its effect in relieving VD by suppressing calcium overload.

Effects of miR-34c-5p on Sodium, Potassium, and Calcium Channel Currents in C2C12 Myotubes.

The aim of this study was to investigate the effects of miR-34c-5p on the main voltage-dependent ion channels in skeletal muscle cells. This study focused on the effects of miR-34c-5p on sodium, potassium, and calcium currents in C2C12 myoblasts. The miR-34c-5p overexpression group, knockdown group, and control group were differentiated for 7days, fused into myotubes, and used for the whole-cell patch clamp recording. Compared with the control group, the whole-cell sodium current density of the other two groups had no significant changes. In the knockdown group, the delayed rectifier potassium current density was increased (statistically significant), and the whole-cell calcium channel current density did not change. In the overexpression group, the change of rectifier potassium current density was not obvious, while the peak calcium channel current density increased (- 9.23 ± 0.95pA/pF, n = 6 cells for the overexpression group vs. - 6.48 ± 0.64pA/pF, n = 7 cells for the control; p < 0.05). Changes in the expression of miR-34c-5p can affect the electrophysiological characteristics of calcium and potassium voltage-gated channels in C2C12 myotubes. Overexpression of miR-34c-5p increased whole-cell L-type calcium channel current (ICa,L), while miR-34c-5p knockdown increased whole-cell delayed rectifier potassium current (IKd).

Pharmacological Preconditioning Using Diazoxide Regulates Store-Operated Ca2 + Channels in Adult Rat Cardiomyocytes.

Voltage-dependent Ca2+ channels and store-operated Ca2+ channels (SOCs) are the major routes of Ca2+ entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca2+, respectively. Ca2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K+ channel blocker), an intracellular mitochondrial energizing solution, or Ni2+ [a blocker of sodium–calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca2+ with a fluorescent Ca2+ indicator. These results indicate that influx of Ca2+ through SOCs is inhibited by their ROS and Ca2+-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca2+. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia–reperfusion.

Enhanced T-type calcium channel 3.2 activity in sensory neurons contributes to neuropathic-like pain of monosodium iodoacetate-induced knee osteoarthritis.

The monosodium iodoacetate knee osteoarthritis model has been widely used for the evaluation of osteoarthritis pain, but the pathogenesis of associated chronic pain is not fully understood. The T-type calcium channel 3.2 (CaV3.2) is abundantly expressed in the primary sensory neurons, in which it regulates neuronal excitability at both the somata and peripheral terminals and facilitates spontaneous neurotransmitter release at the spinal terminals. In this study, we investigated the involvement of primary sensory neuron-CaV3.2 activation in monosodium iodoacetate osteoarthritis pain. Knee joint osteoarthritis pain was induced by intra-articular injection of monosodium iodoacetate (2 mg) in rats, and sensory behavior was evaluated for 35 days. At that time, knee joint structural histology, primary sensory neuron injury, and inflammatory gliosis in lumbar dorsal root ganglia, and spinal dorsal horn were examined. Primary sensory neuron-T-type calcium channel current by patch-clamp recording and CaV3.2 expression by immunohistochemistry and immunoblots were determined. In a subset of animals, pain relief by CaV3.2 inhibition after delivery of CaV3.2 inhibitor TTA-P2 into sciatic nerve was investigated. Knee injection of monosodium iodoacetate resulted in osteoarthritis histopathology, weight-bearing asymmetry, sensory hypersensitivity of the ipsilateral hindpaw, and inflammatory gliosis in the ipsilateral dorsal root ganglia, sciatic nerve, and spinal dorsal horn. Neuronal injury marker ATF-3 was extensively upregulated in primary sensory neurons, suggesting that neuronal damage was beyond merely knee-innervating primary sensory neurons. T-type current in dissociated primary sensory neurons from lumbar dorsal root ganglia of monosodium iodoacetate rats was significantly increased, and CaV3.2 protein levels in the dorsal root ganglia and spinal dorsal horn ipsilateral to monosodium iodoacetate by immunoblots were significantly increased, compared to controls. Perineural application of TTA-P2 into the ipsilateral sciatic nerve alleviated mechanical hypersensitivity and weight-bearing asymmetry in monosodium iodoacetate osteoarthritis rats. Overall, our findings demonstrate an elevated CaV3.2 expression and enhanced function of primary sensory neuron-T channels in the monosodium iodoacetate osteoarthritis pain. Further study is needed to delineate the importance of dysfunctional primary sensory neuron-CaV3.2 in osteoarthritis pain.

Luteolin suppresses colonic smooth muscle motility via inhibiting L-type calcium channel currents in mice.

As a naturally occurring flavone, luteolin has received much attention due to its antioxidant, anti-inflammatory and anticancer functions. In the present study, we investigated the effect of luteolin on colonic motility and its mechanism using isometric muscle recording and the whole-cell patch-clamp technique in mice. Luteolin dose-dependently inhibited colonic smooth muscles motility and CMMC significantly. BayK8644, an L-type Ca2+ channel agonist, significantly attenuated the luteolin-induced inhibition. Moreover, the calcium currents recorded in colonic smooth muscle cells were dramatically inhibited by luteolin. However, no significant changes were found in the luteolin-induced inhibitory effect in the presence of TEA, a nonselective K+ channel blocker, glibenclamide, an ATP-dependent K+ channel blocker, and apamin, a small-conductance Ca2+-activated K+ channel blocker. Additionally, luteolin did not affect potassium currents. Furthermore, TTX, a Na+ channel blocker, L-NAME, an inhibitor of nitric oxide (NO) synthase, ODQ, an inhibitor of NO-sensitive guanylyl cyclase, and Ani9, a specific ANO1 channels blocker, had no effect on the luteolin-induced suppression. These results suggest that luteolin inhibited colonic smooth muscle motility by inhibiting L-type calcium channels in mice but not through potassium channels, the enteric nervous system (ENS), NO signaling pathways or ANO1 channels of interstitial cells of Cajal (ICCs).

Hydroxysafflor yellow A actives BKCa channels and inhibits L-type Ca channels to induce vascular relaxation

Hydroxy-safflor yellow A (HSYA) can exert a variety of effects upon the vascular system. However, the underlying mechanisms are not clear. The present study is to investigate its vasodilating effect and the mechanisms. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were enrolled for studying effects of HSYA on blood pressure, vasodilation, intracellular Ca2+ transient and membrane ion channels. Vasodilation and intracellular Ca2+ transient were measured by using vasomotor assay and fluorescence imaging system, respectively. The effect of HSYA on the large conductance Ca2+ activated and voltage-gated potassium channel (BKCa channel) currents in rat mesentery artery and on L-type calcium channel (Ca-L) currents in HEK293cells expressed with Ca-L were investigated using patch clamp techniques. Blood pressure of SHR and WKY rats were concentration dependently reduced by HSYA with a larger effect of HSYA in SHR than that in WKY rats. The tension of mesenteric arteries induced by 3 μM phenylephrine was attenuated by HSYA (IC50 = 90.8 μΜ). Patch clamp study showed that HSYA could activate BKCa channels and suppress Ca-L channels in a concentration dependent manner. The results of calcium signaling assays indicated that HSYA could reduce the intracellular free Ca2+ level. These findings demonstrate that HSYA could activate BKCa channels and inhibit Ca-L channels and reduce intracellular free Ca2+ level, which are probably important for its vasodilatory effect.

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We found action potential duration, systolic, and diastolic [Ca2+] to respond most sensitively to changes in L-type calcium channel current. The ryanodine receptor channel cluster structure inside dyadic clefts was found to affect all biomarkers investigated. The shape of clusters observed in experiments by Jayasinghe et al. and channel density within the cluster (characterized by mean occupancy) showed the strongest correlation to the effects on biomarkers.

Cellular Mechanism Underlying rTMS Treatment for the Neural Plasticity of Nervous System in Drosophila Brain.

Repetitive transcranial magnetic stimulation (rTMS) is used as a research tool and clinical treatment for the non-clinical and clinical populations, to modulate brain plasticity. In the case of neurologic and psychiatric disease, there is significant evidence to suggest that rTMS plays an important role in the functional recovery after neurological dysfunction. However, the causal role for rTMS in the recovery of nervous dysfunction remains unclear. The purpose of the present study is to detect the regulation of rTMS on the excitatory neuronal transmission and specify the mode of action of rTMS on the neural plasticity using Drosophila whole brain. Therefore, we identified the effects of rTMS on the neural plasticity of central neural system (CNS) by detecting the electrophysiology properties of projection neurons (PNs) from adult Drosophila brain after rTMS. Using patch clamp recordings, we recorded the mini excitatory postsynaptic current (mEPSC) of PNs after rTMS at varying frequencies (1 Hz and 100 Hz) and intensities (1%, 10%, 50%, and 100%). Then, the chronic electrophysiology recordings, including mEPSC, spontaneous action potential (sAP), and calcium channel currents from PNs after rTMS at low frequency (1 Hz), with low intensity (1%) were detected and the properties of the recordings were analyzed. Finally, the frequency and decay time of mEPSC, the resting potential and frequency of sAP, and the current density and rise time of calcium channel currents were significantly changed by rTMS. Our work reveals that rTMS can be used as a tool to regulate the presynaptic function of neural circuit, by modulating the calcium channel in a frequency-, intensity- and time-dependent manner.