Potassium Channel Currents Research Articles

Scorpion Potassium Channel-blocking Defensin Highlights a Functional Link with Neurotoxin

The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensiiKarsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteusas well as methicillin-resistant Staphylococcus aureus Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50value for the Kv1.3 channel was 510.2 nm Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4 adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins.

The F-Actin Binding Protein TRIOBP-1 Regulates hERG K+ Channels

The voltage-gated potassium channel hERG (human ether-a-go-go-related gene) is the primary pore-forming subunit of the rapidly activating delayed rectifier potassium channel current (IKr) in the heart. The physiological role of cardiac IKr is the repolarization of the ventricular action potential. Although it is known that certain genetic mutations of hERG, or acquired conditions such as hypokalemia, can affect IKr, our understanding of the specific protein-protein interactions underlying hERG regulation are not well known. The cytoplasmic C-terminal portion of hERG was previously used to conduct a yeast two-hybrid screen of a human cardiac library for proteins that could regulate hERG channel function, localization, stability, and relative surface expression. This screen identified TRIOBP-1, an F-actin binding protein associated with cytoskeletal dynamics, as a putative hERG interacting protein. Here, we tested for a direct functional interaction between TRIOBP-1 and hERG channels using a combination of whole-cell patch-clamp electrophysiology, Forster resonance energy transfer (FRET) measurements, and biochemical methods. In a HEK293 cell line stably expressing hERG (HEK293-hERG), expression of TRIOBP-1 reduced hERG mean peak tail current density by 50% relative to pcDNA3 controls. Western blotting of HEK293-hERG cells showed a clear reduction in hERG signal of both the mature fully-glycosylated form destined for the plasma membrane and the immature core-glycosylated form when TRIOBP-1 was expressed. FRET analysis revealed robust FRET between TRIOBP-1-CFP and the cytoplasmic C-terminal portion of hERG (hERG1a#666-1159-Citrine), suggesting a direct and specific interaction between TRIOBP-1 and hERG channels. Expressing TRIOBP-1 in cardiomyocytes derived from human induced pluriopotent stem cells reduced native IKr relative to pcDNA3 controls. Together, these experiments provide evidence that hERG channels and cardiac IKr are regulated by a direct interaction with TRIOBP-1.

Short- and long-term inhibition of cardiac inward-rectifier potassium channel current by an antiarrhythmic drug bepridil.

Bepridil is an effective antiarrhythmic drug on supraventricular and ventricular arrhythmias, and inhibitor of calmodulin. Recent investigations have been elucidating that bepridil exerts antiarrhythmic effects through its acute and chronic application for patients. The aim of this study was to identify the efficacy and the potential mechanism of bepridil on the inward-rectifier potassium channel in neonatal rat cardiomyocytes in acute- and long-term conditions. Bepridil inhibited inward-rectifier potassium current (I K1) as a short-term effect with IC50 of 17 μM. Bepridil also reduced I K1 of neonatal cardiomyocytes when applied for 24h in the culture medium with IC50 of 2.7 μM. Both a calmodulin inhibitor (W-7) and an inhibitor of calmodulin-kinase II (KN93) reduced I K1 when applied for 24h as a long-term effect in the same fashion, suggesting that the long-term application of bepridil inhibits I K1 more potently than that of the short-term application through the inhibition of calmodulin kinase II pathway in cardiomyocytes.

Effect of substance P on the potassium and calcium currents of colonic smooth muscle cells

To investigate the effect of substance P(SP) on the spontaneous contractile activity of smooth muscle cells,the large-conductance calcium-activated potassium channel currents (IBKCa) and the L-type calcium channel currents (ICaL) in rat smooth muscle cells of the proximal colon. A total of 24 healthy male Wista rats were used in this test. The change of smooth muscle strips spontaneous contraction of rat proximal colon after adding SP was recorded by a physiological signal stystem (RM6240). The IBKCa and ICaL were measured via the whole cell patch-clamp technique. The longitudinal muscle contraction was obviously increased concentration-dependently after adding different concentrations of SP (10(-7)-10(-6) mol/L), so as the circular muscle while adding SP(10(-8)-10(-6) mol/L) (all P<0.05). Compared with the control group, IBKCa was decreased after adding SP(10(-6) mol/L). Under the stimulating voltage of 60 mV, the IBKCa current density was (11.71±1.65) pA/pF, which was significantly lower compared with the control group (14.42±2.89) pA/pF (P<0.05). The ICaL) was apparently increased. Under the stimulating voltage of 0 mV, the ICaL) currents density was (-5.04±0.67) pA/pF, compared with the control group (-4.25±0.46) pA/pF, which was significantly increased (P<0.01). SP can promote the spontaneous contractile activity of colon smooth muscle of rats in vitro.And SP decrease IBKCa representatively while apparently increase ICaL). That is probably one of the mechanism SP regulate the gastrointestinal motility.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Epinephrine reversed high-concentration bupivacaine-induced inhibition of calcium channels and transient outward potassium current channels, but not on sodium channel in ventricular myocytes of rats.

BackgroundEpinephrine is a first-line drug for cardiopulmonary resuscitation, but its efficacy in the treatment of bupivacaine-induced cardiac toxicity is still in question. We hypothesized that epinephrine can reverse cardiac inhibition of bupivacaine by modulating ion flows through the ventricular myocyte membrane channels of rats. The aim of this study was to observe and report the effects of epinephrine on high-concentration bupivacaine-induced inhibition of sodium (INa), L-type calcium (ICa-L), and transient outward potassium (Ito) currents in the ventricular myocytes of rats.MethodsThe ventricular myocytes were isolated from Sprague-Dawley rats (250-300 g) by acute enzymatic dissociation. The whole-cell patch clamp technique was used to record the ion channel currents in single ventricular myocytes both before and after administration of medications.ResultAdministration of bupivacaine 100 μmol/L significantly reduced INa, (P < 0.05). However, administration of bupivacaine 100 μmol/L in conjunction with epinephrine 0.15 μg/ml had no effect in restoring INa to its previous state. Similarly, a sharp decline of ICa-L and Ito was observed after administration of bupivacaine 100 μmol/L (P < 0.05). In contrast to INa, ICa-L and Ito were significantly improved after the administration of the aforementioned combination of bupivacaine and epinephrine (P < 0.05).ConclusionEpinephrine can reverse high-concentration bupivacaine induced inhibition of ICa-L and Ito, but not INa. Thus, epinephrine’s effectiveness in reversal of bupivacaine-induced cardiac toxicity secondary to sodium channel inhibition may be limited.

Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD)

To elucidate the role of Zn2+-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia–ischemia, PC12 cells were exposed to Oxygen–Glucose Deprivation (OGD) solution mimicking the hypoxic–ischemic condition in neuron, and the effect of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn2+ chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P<0.01 at 3h, 6h and 24h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1h, but decrease at 3h) and decreased the concentration of intracellular glutamate (P<0.05 at 3h and 6h, P<0.01 at 24h respectively compared to control) and GluR2 expression (P<0.05 at 3h, 6h and 24h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic–ischemic condition.

A Combination of Intravenous Genistein Plus Mg2+ Enhances Antihypertensive Effects in SHR by Endothelial Protection and BKCa Channel Inhibition.

The effects of combining genistein (GST) plus magnesium (Mg) upon the development of hypertension were examined in 28 twelve-week-old male spontaneous hypertension rats (SHRs). Four experimental groups were tested: SHR (0.9% NaCl and DMSO), SHR + GST (0.9% NaCl and GST 5mg/kg/day), SHR + Mg (Mg(2+) 0.75 mmol/kg/day and DMSO), and SHR + GST + Mg (Mg(2+) 0.75 mmol/kg/day and GST 5mg/kg/day). A group of normotensive genetic control, Wistar-Kyoto (WKY) rats were also included for comparison. Drugs were administrated intravenously daily for 30 days. Systolic blood pressure (SBP) and heart rate were measured by tail-cuff plethysmography every five days. Vascular tone of mesenteric arteries was examined by an isometric force transducer. Big-conductance calcium-activated potassium channel (BKCa) currents were detected by whole-cell patch-clamp techniques. SBP in SHRs was significantly elevated vs. that in WKY rats. GST or Mg lowered SBP of SHRs. Their combination enhanced antihypertensive effects, as indicated by significantly lowered SBP and shorter onset times. GST or Mg individually improved endothelial dysfunction of SHRs. However, again their combination enhanced endothelial protection, nearly restoring maximal relaxation responses to those seen in WKY. BKCa currents in SHRs were increased compared with WKY rats. GST, Mg, and their combination restored BKCa currents to those of WKY rats. The combination of GST and Mg produces antihypertensive effects and improvement of endothelial dysfunction, which are substantially greater than that when either is used individually. These results suggest a novel and feasible protocol for the prevention and treatment of hypertension and related cardio- and cerebro-vascular diseases.

ZC88, a novel N-type calcium channel blocker from 4-amino-piperidine derivatives state-dependent inhibits Cav2.2 calcium channels

Small molecular inhibitors of Cav2.2 have been reported for the treatment of neuropathic pain; however, low selectivity and side effects limit their further development. In our study, a series of new compounds were designed and synthesized by optimizing the 4-amino-piperidine template. The results show that ZC88 inhibits transiently expressed Cav2.2 in state-dependent manner in oocytes with an IC50 of 0.45±0.09μM. The steady-state inactivation relationship curve is shifted to more negative potentials for the calcium channels, suggesting that ZC88 blocks inactivated state of the channel. ZC88 does not present any remarkable effects on voltage-gated P/Q-type calcium channel currents, l-type calcium channel currents, potassium channel and sodium channel currents. Taken together, these in vitro data suggest that ZC88 is a voltage-dependent, subtype-selective Cav2.2 channel inhibitor and can achieve an improved therapeutic window over the relatively state-independent Cav2.2-selective inhibitor, which may have potential to be developed into a novel analgesic agent.

Preclinical Evaluation of Orally Bioavailable Small-Molecule Inhibitors of Complement Factor D As a Potential Treatment for Paroxysmal Nocturnal Hemoglobinuria

BACKGROUNDComplement factor D, a serine protease, plays an essential role in the activation of the alternative complement pathway and provides important amplification of the classical and lectin complement pathways. Cleavage of factor B by factor D generates C3 convertase that leads to opsonization of targeted surfaces with complement activation fragments and to the formation of the terminal complement complex (TCC); both events lead to cell lysis. Complement dysregulation underlies multiple hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycophosphatidylinositol-anchored complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antibody eculizumab. Although eculizumab lessens intravascular hemolysis, it does not prevent opsonization of erythrocytes and subsequent extravascular hemolysis by immune cells. In contrast, factor D inhibitors are expected to inhibit both terminal complement pathway activation as well as opsonization and should, therefore, be well-positioned to potentially serve this unmet medical need. Herein, we present the preclinical evaluation of our small-molecule inhibitors of factor D including potency, off-target activities, metabolism, and pharmacokinetic properties.METHODS & RESULTS: Initial inhibitors were discovered through laboratory and virtual screening efforts. A high-resolution (1.5 Å) X-ray structure of an early proprietary inhibitor co-crystallized with factor D aided our optimization campaign that culminated in compounds with IC50 values below 100 nM in biochemical protease assays using natural and non-specific substrates. The potent inhibitory effect of the compounds on factor D protease activity translated to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-digit nM. These small molecules are highly selective for factor D and displayed no significant inhibitory effect on a panel of human serine proteases. In accordance with the high selectivity, the inhibitors showed minimal cellular toxicity, no effect on multiple human receptor-ligand interactions, and no inhibition of hERG potassium channel current. Finally, oral and intravenous administration of selected lead compounds to preclinical animal species showed pharmacokinetic properties that suggest their suitability for oral dosing in humans. CONCLUSIONSWe have discovered highly active small-molecule inhibitors of factor D that demonstrate oral bioavailability and low off-target activities. These key attributes, and the potential to inhibit both intravascular and extravascular hemolysis through inhibition of both terminal complement pathway and opsonization, position these inhibitors as promising development candidates for the oral treatment of PNH. DisclosuresWiles:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

Muscarinic-mediated vasoconstriction in human, rat and sheep umbilical cords and related vasoconstriction mechanisms.

The umbilical cord provides nutrition and oxygen to the fetus. The aim of this study was to determine the effects of acetylcholine (ACh) on umbilical cords from humans and other mammals, and the mechanisms of ACh-mediated vasoconstriction in the human umbilical cord. Human and animal umbilical cords used in vascular and cellular experiments. Institute for Fetology, First Hospital of Soochow University, Suzhou, China. A total of 85 pregnant women, 16 Sprague Dawley rats and seven pregnant sheep. Umbilical cord veins and arteries from humans, rats and sheep, aortas and mesenteric arteries from rats, and mesenteric, carotid and femoral arteries from ovine fetuses were used to compare vascular functions in response to ACh and to determine the mechanisms of ACh-mediated umbilical vasoconstriction. Vascular tension and ion channel currents were measured on isolated vessels and smooth muscle cells from human umbilical cords. Provision of new evidence to conclude that ACh-stimulated vasoconstriction is common to all umbilical cords, and cellular mechanisms are linked to potassium channels. ACh caused reliable vasoconstriction in umbilical veins/arteries in humans, rats and sheep, but not in any other vessels, including fetal vessels. Atropine inhibited the effects of ACh. The mRNA of ACh-muscarinic receptor subtypes M1 -M5 was expressed in human umbilical vessels. The protein kinase C antagonist GF109203X and the calcium inhibitor nifedipine decreased ACh-induced vasoconstriction in human umbilical vessels. ACh also caused a reduction in whole-cell potassium channel currents and the single-channel current of large-conductance calcium-activated potassium (BKca) channels. Umbilical vessels are significantly different from other vessels in their response to ACh. BKca channels in smooth muscle cells may play important roles in ACh-mediated vasoconstriction in human umbilical cords. This information may be important for fetal medicine and practice with regard to the effect on fetal development of umbilical vascular functions.

Chlorogenic acid alters the voltage-gated potassium channel currents of trigeminal ganglion neurons.

Chlorogenic acid (5-caffeoylquinic acid, CGA) is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer's disease and neuropathic pain disorders. However, its mechanism of action is unclear. Voltage-gated potassium channel (Kv) is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,A and IK,V channels. Trigeminal ganglion (TRG) neurons were acutely disassociated from the rat TRG, and two different doses of CGA (0.2 and 1 mmol⋅L−1) were applied to the cells. Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,A and IK,V channels. The results demonstrated that 0.2 mmol⋅L−1 CGA decreased the peak current density of IK,A. Both 0.2 mmol⋅L−1 and 1 mmol⋅L−1 CGA also caused a significant reduction in the activation and inactivation thresholds of IK,A and IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,A and IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.

Cocaine increases dopaminergic neuron and motor activity via midbrain α1 adrenergic signaling.

Cocaine reinforcement is mediated by increased extracellular dopamine levels in the forebrain. This neurochemical effect was thought to require inhibition of dopamine reuptake, but cocaine is still reinforcing even in the absence of the dopamine transporter. Here, we demonstrate that the rapid elevation in dopamine levels and motor activity elicited by cocaine involves α1 receptor activation within the ventral midbrain. Activation of α1 receptors increases dopaminergic neuron burst firing by decreasing the calcium-activated potassium channel current (SK), as well as elevates dopaminergic neuron pacemaker firing through modulation of both SK and the hyperpolarization-activated cation currents (Ih). Furthermore, we found that cocaine increases both the pacemaker and burst-firing frequency of rat ventral-midbrain dopaminergic neurons through an α1 adrenergic receptor-dependent mechanism within the ventral tegmental area and substantia nigra pars compacta. These results demonstrate the mechanism underlying the critical role of α1 adrenergic receptors in the regulation of dopamine neurotransmission and behavior by cocaine.

Exogenous H2 S enhances mice gastric smooth muscle tension through S-sulfhydration of KV 4.3, mediating the inhibition of the voltage-dependent potassium current.

Hydrogen sulfide (H2 S) has been shown to have an excitatory effect on gastric motility, but the underlying molecular mechanism is unclear. In this study, we aimed to investigate the possible targets of H2 S and determine how H2 S affects its target proteins during H2 S-induced contraction. Patch-clamp and potentiometric fluorescence dye were utilized to measure the electrophysiological changes. The Biotin-switch assay was utilized to detect the protein S-sulfhydration. The isometric tension measurement was conducted too. Exogenous H2 S enhanced the tonic contraction of gastric antral smooth muscle, and voltage-dependent potassium channel (KV ) blocker and Dithiothreitol (DTT, a reducing agent) abolished the excitatory effect of NaHS. Exogenous H2 S inhibited the fast inactivation component of the voltage-dependent potassium channel current (IKVfast ) in isolated gastric antral smooth muscle cells. H2 S inhibited the KV 4.3 current in H293 cells with heterologous expression of KV 4.3, but did not inhibit the KV 4.1 and KV 4.2 currents, which together contribute greatly to IKVfast . NaHS significantly decreased the membrane potential in cultured gastric smooth muscle cells, but the NaHS-induced depolarization was suppressed by knockdown of KV 4.3 and N-ethylamaleimide (NEM), a free thiol group blocker. In addition, NaHS sulfhydrated KV 4.3 in H293 cells and in gastric smooth muscle tissue. However, this S-sulfhydration was inhibited by NEM and DTT. Meanwhile the NaHS-induced inhibition of IKVfast and KV 4.3 was also blocked by NEM and DTT. These results suggest that exogenous H2 S sulfhydrates KV 4.3 to decrease the membrane potential, thereby enhancing the basal tension of gastric antral smooth muscle.

Abstract 201: Overexpression of Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Potentiates Angiotensin II (AngII)-Mediated Signaling in Neurons

AngII signaling in the brain regulates systemic cardiovascular function. AngII modulates neuronal ion channel activity and neuronal firing via reactive oxygen species (ROS) and redox-sensitive proteins. Our recent studies indicate that in neurons AngII increases activity (i.e. phosphorylation) of CaMKII, a signaling intermediate known to inhibit potassium (K+) channel current. However, the potential cross-talk between AngII, ROS, and CaMKII in modulating neuronal K+ channel activity and the in vivo central AngII-induced pressor response remains unclear. Here, we tested the hypothesis that CaMKII overexpression exacerbates the AngII-dependent inhibition of K+ channel current and the acute central AngII-induced increase in blood pressure. The neuron specific isoform of CaMKII (CaMKIIα) was cloned, and CaMKIIα adenovirus was generated (AdCaMKIIα). Adenovirus-mediated overexpression of total and active CaMKII protein were assessed in differentiated mouse catecholaminergic (CATH.a) neurons by measuring total and phosphorylated CaMKII protein levels, respectively, via Western blot analysis. To investigate the effect of CaMKIIα overexpression on K+ channel activity, CATH.a neurons were transduced with AdCaMKIIα or control adenovirus, AdEmpty, and voltage-gated K+ current (IKV) was measured by the whole cell patch-clamp technique. Baseline IKV was measured followed by 5 mins of AngII (100 nM) superfusion. AngII lowered steady-state current (ISS) and peak current (Ipeak) in AdEmpty-transduced neurons (ISS by 20 ± 4%; Ipeak by 19 ± 3%), and this inhibitory response was significantly potentiated in AdCaMKIIα-transduced neurons (ISS by 40 ± 7%; Ipeak by 41 ± 5%; p&lt; 0.05). Preliminary in vivo studies suggest that the central AngII-induced pressor response is exacerbated in mice intracerebroventricularly injected with AdCaMKIIα as compared to control virus-injected mice. These data indicate that CaMKIIα overexpression potentiates AngII-mediated inhibition of IKv and the central AngII-induced pressor response. Future studies will investigate the ROS-mediated post-translational modifications of CaMKIIα in AngII-stimulated neurons and the contribution of these modifications to ion channel activity and blood pressure regulation.

A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs).

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.

Antiarrhythmic efficacy of CPUY102122, a multiple ion channel blocker, on rabbits with ischemia/reperfusion injury

BackgroundThe antiarrhythmic potential of a novel multichannel blocker CPUY102122 (CY22) was investigated in the present study. MethodsThe effect of CY22 on rapid delayed rectifier potassium channel current (IKr) was studied using whole-cell patch clamp techniques in Chinese Hamster Ovary cells stably expressing human Ether-à-go-go-Related Gene. We further evaluated the antioxidant effects of CY22 and demonstrated the reversal of connexin down-regulation in the development of cardiac ventricular arrhythmias, which was produced using coronary ligation/reperfusion in rabbits. CY22 and Amiodarone were administered 30min prior to the procedure. Next, electrocardiograms were recorded, protein expression of left ventricular Connexin43 (Cx43), non-phosphorylation-Cx43 (np-Cx43), Rac-1 and gp-91[phox] were assayed using Western blot analysis, microstructural changes in the myocardium were observed and redox system activity was assayed. ResultsCY22 inhibited IKr in a concentration-dependent manner with IC50 value of 2.8±0.8μmol/L. CY22 treatment significantly decreased T-wave amplitude and QTc arrhythmic scores and ameliorated the shape of the infarcted myocardium compared to the model group. CY22 decreased the serum levels of creatine kinase, lactate dehydrogenase, and myocardial levels of malondialdehyde, as well as increased superoxide dismutase activity. Cx43 expression in the left ventricle was significantly increased by CY22 treatment, which significantly decreased np-43 expression, Rac-1 activity and gp-91[phox] protein expression. ConclusionsThese results indicated that CY22 has both antiarrhythmic and cardiovascular protective effects partly by blocking IKr, the production of antioxidants and protection of Cx43.

Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries.

The mechanisms underlying developmental programming are poorly understood but may be associated with adaptations by the fetus in response to changes in the maternal environment during pregnancy. We hypothesized that maternal nutrient restriction during pregnancy alters vasodilator responses in fetal coronary arteries. Pregnant ewes were fed a control [100% U.S. National Research Council (NRC)] or nutrient-restricted (60% NRC) diet from days 50 to 130 of gestation (term = 145 days); fetal tissues were collected at day 130. In coronary arteries isolated from control fetal lambs, relaxation to bradykinin was unaffected by nitro-l-arginine (NLA). Iberiotoxin or contraction with KCl abolished the NLA-resistant response to bradykinin. In fetal coronary arteries from nutrient-restricted ewes, relaxation to bradykinin was fully suppressed by NLA. Large-conductance, calcium-activated potassium channel (BKCa) currents did not differ in coronary smooth muscle cells from control and nutrient-restricted animals. The BKCa openers, BMS 191011 and NS1619, and 14,15-epoxyeicosatrienoic acid [a putative endothelium-derived hyperpolarizing factor (EDHF)] each caused fetal coronary artery relaxation and BKCa current activation that was unaffected by maternal nutrient restriction. Expression of BKCa-channel subunits did not differ in fetal coronary arteries from control or undernourished ewes. The results indicate that maternal undernutrition during pregnancy results in loss of the EDHF-like pathway in fetal coronary arteries in response to bradykinin, an effect that cannot be explained by a decreased number or activity of BKCa channels or by decreased sensitivity to mediators that activate BKCa channels in vascular smooth muscle cells. Under these conditions, bradykinin-induced relaxation is completely dependent on nitric oxide, which may represent an adaptive response to compensate for the absence of the EDHF-like pathway.

Far red/near infrared light induces vasodilation and an increase in activity of large conductance potassium channel currents in murine femoral arterial muscle (677.13)

Over 8 million Americans are treated for symptoms related to peripheral artery disease (PAD), such as intermittent claudication, impaired wound healing, and critical limb ischemia. We have shown light irradiation at the far red/near infrared region (670 nm (R/NIR)) can stimulate angiogenesis by increasing NO independent of NOS through release of NO from nitrosyl‐heme stores. We hypothesized R/NIR can stimulate vasodilation through increases in large conductance potassium channel (BKCa) activity. To test vasodilation, facial arteries from C57Bl/6 mice were isolated, pressurized to 60mmHg, and pre‐constricted with U46619 in the dark.The vessels were irradiated with 670 nm light (10mW/cm2) up to 5 min at a time with 10 min dark period between irradiation. Vessel diameter increased up to 17.8% (±3.0, p&lt;0.05) after 5 min of R/NIR exposure. The effect of red light on single channel BKCa current was determined in enzymatically dissociated murine femoral arterial muscle cells, using the patch clamp technique and symmetrical KCl [145mM] recording solution at a patch potential of +60 mV (RT). Exposure of cell attached patches to light ( λ =670 nm, 10 mW/cm2) markedly increased the opening frequency of the large conductance potassium channels (BKCa) single channel current. This effect of light on BKCa single channel current activity could be the underlying mechanism for the vasodilatory action of red light in isolated murine femoral arterial segments. Conclusion: R/NIR exposure acts to stimulate vasodilation, by increasing the opening frequency of BKCa single channel current recorded in cell attached patches of femoral arterial smooth muscle cells.

Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis.

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.