Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function.

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Attenuation of the extracellular matrix increases the number of synapses but suppresses synaptic plasticity through upregulation of SK channels.

The effect of brain extracellular matrix (ECM) on synaptic plasticity remains controversial. Here, we show that targeted enzymatic attenuation with chondroitinase ABC (ChABC) of ECM triggers the appearance of new glutamatergic synapses on hippocampal pyramidal neurons, thereby increasing the amplitude of field EPSPs while decreasing both the mean miniature EPSC amplitude and AMPA/NMDA ratio. Although the increased proportion of 'unpotentiated' synapses caused by ECM attenuation should promote long-term potentiation (LTP), surprisingly, LTP was suppressed. The upregulation of small conductance Ca2+-activated K+ (SK) channels decreased the excitability of pyramidal neurons, thereby suppressing LTP. A blockade of SK channels restored cell excitability and enhanced LTP; this enhancement was abolished by a blockade of Rho-associated protein kinase (ROCK), which is involved in the maturation of dendritic spines. Thus, targeting ECM elicits the appearance of new synapses, which can have potential applications in regenerative medicine. However, this process is compensated for by a reduction in postsynaptic neuron excitability, preventing network overexcitation at the expense of synaptic plasticity.

Bupivacaine inhibits a small conductance calcium-activated potassium type 2 channel in human embryonic kidney 293 cells.

BACKGROUND: Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. METHODS: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration-response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. RESULTS: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. CONCLUSIONS: The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

SK channel blockade prevents hypoxia-induced ventricular arrhythmias through inhibition of Ca2+/voltage uncoupling in hypertrophied hearts.

Ventricular arrhythmia (VA) is the major cause of death in patients with left ventricular (LV) hypertrophy and/or acute ischemia. We hypothesized that apamin, a blocker of small-conductance Ca2+-activated K+ (SK) channels, alters Ca2+ handling and exhibits anti-arrhythmic effects in ventricular myocardium. Spontaneous hypertensive rats were used as a model of LV hypertrophy. A dual optical mapping of membrane potential (Vm) and intracellular calcium (Cai) was performed during global hypoxia (GH) on the Langendorff perfusion system. The majority of pacing-induced VAs during GH were initiated by triggered activities. Pretreatment of apamin (100 nmol/L) significantly inhibited the VA inducibility. Compared with SK channel blockers (apamin and NS8593), non-SK channel blockers (glibenclamide and 4-AP) did not exhibit anti-arrhythmic effects. Apamin prevented not only action potential duration (APD80) shortening (-18.7 [95% confidence interval, -35.2 to -6.05] ms vs. -2.75 [95% CI, -10.45 to 12.65] ms, P = 0.04) but also calcium transient duration (CaTD80) prolongation (14.52 [95% CI, 8.8-20.35] ms vs. 3.85 [95% CI, -3.3 to 12.1] ms, P < 0.01), thereby reducing CaTD80 - APD80, which denotes "Cai/Vm uncoupling" (33.22 [95% CI, 22-48.4] ms vs. 6.6 [95% CI, 0-14.85] ms, P < 0.01). The reduction of Cai/Vm uncoupling was attributable to less prolonged Ca2+ decay constant and suppression of diastolic Cai increase by apamin. The inhibition of VA inducibility and changes in APs/CaTs parameters caused by apamin was negated by the addition of ouabain, an inhibitor of Na+/K+ pump. Apamin attenuates APD shortening, Ca2+ handling abnormalities, and Cai/Vm uncoupling, leading to inhibition of VA occurrence in hypoxic hypertrophied hearts.NEW & NOTEWORTHY We demonstrated that hypoxia-induced ventricular arrhythmias were mainly initiated by Ca2+-loaded triggered activities in hypertrophied hearts. The blockades of small-conductance Ca2+-activated K+ channels, especially "apamin," showed anti-arrhythmic effects by alleviation of not only action potential duration shortening but also Ca2+ handling abnormalities, most notably the "Ca2+/voltage uncoupling."

Development of pyrene-based fluorescent ether lipid as inhibitor of SK3 ion channels.

We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 µM) and by its inhibition of SK3-dependent cancer cell migration at (-32.5% ± 4.8% at 1 µM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.

Simultaneous activation of the small conductance calcium-activated potassium current by acetylcholine and inhibition of sodium current by ajmaline cause J-wave syndrome in Langendorff-perfused rabbit ventricles.

BACKGROUND: Concomitant apamin-sensitive small conductance calcium-activated potassium current (IKAS) activation and sodium current inhibition induce J-wave syndrome (JWS) in rabbit hearts. Sudden death in JWS occurs predominantly in men at night when parasympathetic tone is strong. OBJECTIVE: The purpose of this study was to test the hypotheses that acetylcholine (ACh), the parasympathetic transmitter, activates IKAS and causes JWS in the presence of ajmaline. METHODS: We performed optical mapping in Langendorff-perfused rabbit hearts and whole-cell voltage clamp to determine IKAS in isolated ventricular cardiomyocytes. RESULTS: ACh (1 µM) + ajmaline (2 µM) induced J-point elevations in all (6 male and 6 female) hearts from 0.01± 0.01 to 0.31 ± 0.05 mV (P<.001), which were reduced by apamin (specific IKAS inhibitor, 100 nM) to 0.14 ± 0.02 mV (P<.001). More J-point elevation was noted in male than in female hearts (P=.037). Patch clamp studies showed that ACh significantly (P<.001) activated IKAS in isolated male but not in female ventricular myocytes (n=8). Optical mapping studies showed that ACh induced action potential duration (APD) heterogeneity, which was more significant in right than in left ventricles. Apamin in the presence of ACh prolonged both APD at the level of 25% (P<.001) and APD at the level of 80% (P<.001) and attenuated APD heterogeneity. Ajmaline further increased APD heterogeneity induced by ACh. Ventricular arrhythmias were induced in 6 of 6 male and 1 of 6 female hearts (P=.015) in the presence of ACh and ajmaline, which was significantly suppressed by apamin in the former. CONCLUSION: ACh activates ventricular IKAS. ACh and ajmaline induce JWS and facilitate the induction of ventricular arrhythmias more in male than in female ventricles.

Ablation of small conductance calcium-activated potassium type-2 channel (SK2) delays occurrence of bupivacaine-induced cardiotoxicity in isolated mouse hearts.

Bupivacaine is frequently used for conducting regional anesthesia. When accidentally injected or excessively absorbed into circulation, bupivacaine can induce severe arrhythmia and potentially lead to cardiac arrest. The specific mechanisms underlying this cardiotoxicity, however, remain to be clarified. We transfected HEK-293 cells to express the small conductance calcium-activated potassium type-2 channel (SK2), and used a whole-cell patch clamp method in order to explore how bupivacaine affected these channels. We subsequently used SK2 knockout mice to explore the relevance of SK2 channels in bupivacaine-induced cardiotoxicity in isolating mouse hearts, mounting them on a Langendorff apparatus, and perfusing them with bupivacaine. Using this system, arrhythmia, asystole, and cardiac functions were monitored. We observed dose-dependent inhibition of SK2 channels by bupivacaine: half-maximal inhibitory concentration (IC50) value = 18.6 µM (95% CI 10.8-32.1). When SK2 knockout (SK2 -/-) or wild-type (WT) mice were perfused with Krebs-Henseleit buffer (KHB), we did not observe any instances of arrhythmia. When SK2 -/- mice or WT were perfused with KHB containing bupivacaine (40 µM), the time to arrhythmia (Tarrhythmia) and time to asystole (Tasystole) were both significantly longer in SK2 -/- mice relative to WT mice (P < 0.001). Similarly, SK2 -/- mice exhibited a significantly longer time to 25%, 50%, and 75% reductions in heart rate (HR) and rate-pressure product (RPP) relative to WT mice following bupivacaine perfusion (P < 0.001). These results reveal that bupivacaine was able to mediate a dose-dependent inhibition of SK2 channels in HEK-293 cells, and deletion of SK2 channels can delay bupivacaine-induced cardiotoxicity in isolated mouse hearts.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

Hypoxia Promotes Prostate Cancer Aggressiveness by Upregulating EMT-Activator Zeb1 and SK3 Channel Expression.

Hypoxia is a well-established feature of prostate cancer (PCa) and is associated with disease aggressiveness. The hypoxic microenvironment initiates multiple adaptive responses including epithelial-to-mesenchymal transition (EMT) and a remodeling of calcium homeostasis involved in cancer progression. In the present study, we identified a new hypoxia signaling pathway with a positive feedback loop between the EMT transcription factor Zeb1 and SK3, a Ca2+-activated K+ channel, which leads to amplifying store-operated Ca2+ entry. Zeb1 and SK3 channel were strongly upregulated by hypoxia both in vitro and ex vivo in organotypic cultures of human PCa. Taking into account the sensitivity of the SK3 channel to the membrane lipid composition, we identified lipids such as Ohmline (an alkyl ether lipid and SK3 inhibitor), linoleic acid (LA) and eicosapentaenoic acid (EPA) (fatty acids associated with indolent PCa), which were able to completely abrogate the hypoxia-induced changes in Zeb1 expression. Ultimately, better understanding of this new hypoxia-induced EMT pathway may allow to develop adjuvant therapeutic strategies, in order to control PCa aggressiveness and improve treatment outcomes.

The Small Conductance Calcium-Activated Potassium Channel Inhibitors NS8593 and UCL1684 Prevent the Development of Atrial Fibrillation Through Atrial-Selective Inhibition of Sodium Channel Activity.

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Kcnn2 blockade reverses learning deficits in a mouse model of fetal alcohol spectrum disorders.

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.

Small and intermediate Ca2+-sensitive K+ channels do not play a role in vascular conductance during resting blood flow in the anaesthetised pig.

Flow-induced dilation in resistance arteries is mediated by endothelium-dependent hyperpolarisation via small and intermediate conducting Ca2+ sensitive K+ channels. The aim of the current study was to assess the effect of blocking both channels, using the toxins apamin and charybdotoxin, on flow-induced dilation in a conduit artery and vascular conductance. Experiments were carried out on the iliac and its vascular bed in anaesthetised pigs (n = 4). Flow-induced dilation and vascular conductance (∆F/∆P) were assessed before and after administration of toxins intra-arterially (i.a.) at 50 µg kg-1. Iliac diameter increased from baseline to 2.39 ± 0.4 mm before and 2.09 ± 0.46 mm after toxin administration, which was not significantly different (P = 0.63, Student's paired t test). Control conductance was 1.49 ± 0.27 ml min-1 mmHg-1 (P < 0.00001, ANOVA), and 1.53 ± 0.18 ml min-1 mmHg-1 (P < 0.00001, ANOVA) in the presence of the toxins which was not significantly different (P = 0.93 homogeneity of regression analysis). There was a small but significant increase in mean arterial pressure after the toxins were administered, from 74 ± 5 to 80 ± 9 mmHg (P = 0.03, Student's paired t test); but all other measured parameters were not significantly affected. Small- and intermediate-conducting Ca2+-sensitive K+ channels are not involved in flow-mediated dilation in conduit arteries and do not play a role in resistance vessel diameter maintenance at resting blood flow.

Apamin Improves Prefrontal Nicotinic Impairment in Mouse Model of Alzheimer's Disease.

Disruption of attention is an early and disabling symptom of Alzheimer's disease (AD). The underlying cellular mechanisms are poorly understood and treatment options for patients are limited. These early attention deficits are evident in the TgCRND8 mouse, a well-established murine model of AD that recapitulates several features of the disease. Here, we report severe impairment of the nicotinic receptor-mediated excitation of prefrontal attentional circuitry in TgCRND8 mice relative to wild-type littermate controls. We demonstrate that this impairment can be remedied by apamin, a bee venom neurotoxin peptide that acts as a selective antagonist to the SK family of calcium-sensitive potassium channels. We probe this seeming upregulation of calcium-sensitive inhibition and find that the attenuated nicotinic firing rates in TgCRND8 attention circuits are mediated neither by greater cellular calcium signals nor by elevated SK channel expression. Instead, we find that TgCRND8 mice show enhanced functional coupling of nicotinic calcium signals to inhibition. This SK-mediated inhibition exerts a powerful negative feedback on nicotinic excitation, dampening attention-relevant signaling in the TgCRND8 brain. These mechanistic findings identify a new cellular target involved in the modulation of attention and a novel therapeutic target for early attention deficits in AD.

Impaired Reliability and Precision of Spiking in Adults But Not Juveniles in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common source of intellectual disability and autism. Extensive studies have been performed on the network and behavioral correlates of the syndrome, but our knowledge about intrinsic conductance changes is still limited. In this study, we show a differential effect of FMRP knockout in different subsections of hippocampus using whole-cell patch clamp in mouse hippocampal slices. We observed no significant change in spike numbers in the CA1 region of hippocampus, but a significant increase in CA3, in juvenile mice. However, in adult mice we see a reduction in spike number in the CA1 with no significant difference in CA3. In addition, we see increased variability in spike numbers in CA1 cells following a variety of steady and modulated current step protocols. This effect emerges in adult mice (8 weeks) but not juvenile mice (4 weeks). This increased spiking variability was correlated with reduced spike number and with elevated AHP. The increased AHP arose from elevated SK currents (small conductance calcium-activated potassium channels), but other currents involved in medium AHP, such as Ih and M, were not significantly different. We obtained a partial rescue of the cellular variability phenotype when we blocked SK current using the specific blocker apamin. Our observations provide a single-cell correlate of the network observations of response variability and loss of synchronization, and suggest that the elevation of SK currents in FXS may provide a partial mechanistic explanation for this difference.

MiR-652-3p promotes bladder cancer migration and invasion by targeting KCNN3.

OBJECTIVE: Increasing evidence indicated that microRNAs (miRNAs) are crucial regulators for cancer development. Bladder cancer (BCa) is a major threat to human health. The aim of this study was to analyze the roles of miR-652-3p in BCa, and to explore the associated mechanisms. MATERIALS AND METHODS: MiR-652-3p expression in BCa cell lines was explored using Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) method. MiR-652-3p expression level in BCa tissues was explored at StarBase. Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and transwell invasion assay were conducted to investigate the biological roles of miR-652-3p. The underlying mechanisms of miR-652-3p in NSCLC were investigated using luciferase activity reporter assay and rescue experiments. RESULTS: We showed that miR-652-3p expression level was upregulated in both BCa tissues and cell lines. The knockdown of miR-652-3p significantly inhibited BCa cell proliferation, migration, and invasion in vitro. Moreover, we showed that potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 (KCNN3) was a functional target for miR-652-3p. Besides, the expression of KCNN3 in BCa tissues was negatively correlated with miR-652-3p. CONCLUSIONS: Collectively, these results showed that miR-652-3p could promote BCa cell proliferation, migration, and invasion via directly regulating KCNN3, which may provide a novel therapeutic target for BCa treatment.

Arrhythmia development during inhibition of small-conductance calcium-activated potassium channels in acute myocardial infarction in a porcine model.

AIMS: Acute myocardial infarction (AMI) is associated with intracellular Ca2+ build-up. In healthy ventricles, small conductance Ca2+-activated K+ (SK) channels are present but do not participate in repolarization. However, SK current is increased in chronic myocardial infarction and heart failure, and recently, SK channel inhibition was demonstrated to reduce arrhythmias in AMI rats. Hence, we hypothesized that SK channel inhibitors (NS8593 and AP14145) could reduce arrhythmia development during AMI in a porcine model. METHODS AND RESULTS: Twenty-seven pigs were randomized 1:1:1 to control, NS8593, or AP14145. Haemodynamic and electrophysiological parameters [electrocardiogram (ECG) and monophasic action potentials (MAP)] were continuously recorded. A balloon was placed in the mid-left anterior descending artery, blinded to treatment. Infusion lasted from 10 min before occlusion until 30 min after. Occlusion was maintained for 1 h, followed by 2 h of reperfusion. Upon occlusion, cardiac output dropped similarly in all groups, while blood pressure remained stable. Heart rate decreased in the NS8593 and AP14145 groups. QRS duration increased upon occlusion in all groups but more prominently in AP14145-treated pigs. Inhibition of SK channels did not affect QT interval. Infarct MAP duration shortened comparably in all groups. Ventricular fibrillation developed in 4/9 control-, 4/9 AP14145-, and 2/9 NS8593-treated pigs. Ventricular tachycardia was rarely observed in either group, whereas ventricular extrasystoles occurred comparably in all groups. CONCLUSION: Inhibition of SK channels was neither beneficial nor detrimental to ventricular arrhythmia development in the setting of AMI in this porcine model.

Benzamil inhibits neuronal and heterologously expressed small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of pyramidal neurons in the neocortex and hippocampal formation, where they participate in the local regulation of membrane excitability and synaptic signals. Through their inter-play with Ca2+ channels, SK channels regulate Ca2+ influx triggered by back-propagating action potentials in dendrites. Inhibition of SK channels affects both the amplitude and duration of Ca2+ transients, but the role of Ca2+ clearance mechanisms and their link to SK channel activity has not been established. Here we report the effect of the Na+/Ca2+ exchanger (NCX) inhibitor benzamil on Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased the duration and amplitude of dendritic Ca2+ transients elicited by back-propagating action potentials in hippocampal pyramidal neurons. This data is consistent with previous studies with SK channel blockers and suggests that benzamil inhibits SK channels in addition to the Na+/Ca2+ exchanger. Here we show that indeed both the neuronal SK-mediated IAHP current and the currents mediated by heterologously expressed SK channels were inhibited by benzamil. The inhibition of recombinant SK channels was seen with different K+ concentration gradients, and was stronger at negative voltages. The suppression of SK channels by benzamil is consistent with previous findings on the modulation of Ca2+ signals by SK channels in neurons. We additionally show that benzamil inhibits neuronal voltage-gated calcium currents. The results prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.

Targeting the potassium ion channel genes SK and SH as a novel approach for control of insect pests: efficacy and biosafety.

BACKGROUND: Potassium ion channels play a critical role in the generation of electrical signals and thus provide potential targets for control of insect pests by RNA interference. RESULTS: Genes encoding the small conductance calcium-activated potassium channel (SK) and the voltage-gated potassium channel (SH) were knocked down in Tribolium castaneum by injection and oral delivery of dsRNA (dsTcSK and dsTcSH, respectively). Irrespective of the delivery mechanism a dose-dependent effect was observed for knockdown (KD) of gene expression and insect mortality for both genes. Larvae fed a 400 ng dsRNA mg-1 diet showed significant gene (P < 0.05) knockdown (98% and 83%) for SK and SH, respectively, with corresponding mortalities of 100% and 98% after 7 days. When injected (248.4 ng larva-1 ), gene KD was 99% and 98% for SK and SH, causing 100% and 73.4% mortality, respectively. All developmental stages tested (larvae, early- and late-stage pupae and adults) showed an RNAi-sensitive response for both genes. LC50 values were lower for SK than SH, irrespective of delivery method, demonstrating that the knockdown of SK had a greater effect on larval mortality. Biosafety studies using adult honeybee Apis mellifera showed that there were no significant differences either in expression levels or mortality of honeybees orally dosed with dsTcSK and dsTcSH compared to control-fed bees. Similarly, there was no significant difference in the titre of deformed wing virus, used as a measure of immune suppression, between experimental and control bees. CONCLUSION: This study demonstrates the potential of using RNAi targeting neural receptors as a technology for the control of T. castaneum. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.