Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. METHODS: Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). RESULTS: ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

The therapeutic potential of bee venom-derived Apamin and Melittin conjugates in cancer treatment: A systematic review.

The therapeutic potential of bee venom-derived peptides, particularly apamin and melittin, in cancer treatment has garnered significant attention as a promising avenue for advancing oncology. This systematic review examines preclinical studies highlighting the emerging role of these peptides in enhancing cancer therapies. Melittin and apamin, when conjugated with other therapeutic agents or formulated into novel delivery systems, have demonstrated improved efficacy in targeting tumor cells. Key findings indicate that melittin-based conjugates, such as polyethylene glycol (PEG)ylated versions, show potential in enhancing therapeutic outcomes and minimizing toxicity across various cancer models. Similarly, apamin-conjugated formulations have improved the efficacy of established anti-cancer drugs, contributing to enhanced targeting and reduced systemic toxicity. These developments underscore a growing interest in leveraging bee venom-derived peptides as adjuncts in cancer therapy. The integration of these peptides into treatment regimens offers a promising strategy to address current limitations in cancer treatment, such as drug resistance and off-target effects. However, comprehensive validation through clinical trials is essential to confirm their safety and effectiveness in human patients. This review highlights the global emergence of bee venom-derived peptides in cancer treatment, advocating for continued research and development to fully realize their therapeutic potential.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Protective Effects of Apamin on Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose can cause severe liver damage, but therapeutic options are limited. Apamin is a natural peptide present in bee venom and has antioxidant and anti-inflammatory properties. Accumulating evidence suggests that apamin has favorable actions in rodent models of inflammatory disorders. Here, we examined the effect of apamin on APAP-evoked hepatotoxicity. Intraperitoneal administration of apamin (0.1 mg/kg) alleviated histological abnormalities and reduced serum levels of liver enzymes in mice injected with APAP. Apamin inhibited oxidative stress through an increase in the amount of glutathione and activation of the antioxidant system. Apamin also attenuated apoptosis with inhibition of caspase-3 activation. Moreover, apamin reduced serum and hepatic levels of cytokines in APAP-injected mice. These effects were accompanied by suppression of NF-κB activation. Furthermore, apamin inhibited chemokine expression and inflammatory cell infiltration. Our results suggest that apamin dampens APAP-evoked hepatotoxicity through inhibiting oxidative stress, apoptosis, and inflammation.

Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes.

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Effects of Apamin on MPP+-Induced Calcium Overload and Neurotoxicity by Targeting CaMKII/ERK/p65/STAT3 Signaling Pathways in Dopaminergic Neuronal Cells.

Parkinson's disease (PD), a neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons. The pathogenesis of PD is associated with several factors including oxidative stress, inflammation, and mitochondrial dysfunction. Ca2+ signaling plays a vital role in neuronal signaling and altered Ca2+ homeostasis has been implicated in many neuronal diseases including PD. Recently, we reported that apamin (APM), a selective antagonist of the small-conductivity Ca2+-activated K+ (SK) channel, suppresses neuroinflammatory response. However, the mechanism(s) underlying the vulnerability of DA neurons were not fully understood. In this study, we investigated whether APM affected 1-methyl-4-phenyl pyridinium (MPP+)-mediated neurotoxicity in SH-SY5Y cells and rat embryo primary mesencephalic neurons. We found that APM decreased Ca2+ overload arising from MPP+-induced neurotoxicity response through downregulating the level of CaMKII, phosphorylation of ERK, and translocation of nuclear factor NFκB/signal transducer and activator of transcription (STAT)3. Furthermore, we showed that the correlation of MPP+-mediated Ca2+ overload and ERK/NFκB/STAT3 in the neurotoxicity responses, and dopaminergic neuronal cells loss, was verified through inhibitors. Our findings showed that APM might prevent loss of DA neurons via inhibition of Ca2+-overload-mediated signaling pathway and provide insights regarding the potential use of APM in treating neurodegenerative diseases.

Optimized Apamin-Mediated Nano-Lipidic Carrier Potentially Enhances the Cytotoxicity of Ellagic Acid against Human Breast Cancer Cells.

Ellagic acid has recently attracted increasing attention regarding its role in the prevention and treatment of cancer. Surface functionalized nanocarriers have been recently studied for enhancing cancer cells' penetration and achieving better tumor-targeted delivery of active ingredients. Therefore, the present work aimed at investigating the potential of APA-functionalized emulsomes (EGA-EML-APA) for enhancing cytototoxic activity of EGA against human breast cancer cells. Phospholipon® 90 G: cholesterol molar ratio (PC: CH; X1, mole/mole), Phospholipon® 90 G: Tristearin weight ratio (PC: TS; X2, w/w) and apamin molar concentration (APA conc.; X3, mM) were considered as independent variables, while vesicle size (VS, Y1, nm) and zeta potential (ZP, Y2, mV) were studied as responses. The optimized formulation with minimized vs. and maximized absolute ZP was predicted successfully utilizing a numerical technique. EGA-EML-APA exhibited a significant cytotoxic effect with an IC50 value of 5.472 ± 0.21 µg/mL compared to the obtained value from the free drug 9.09 ± 0.34 µg/mL. Cell cycle profile showed that the optimized formulation arrested MCF-7 cells at G2/M and S phases. In addition, it showed a significant apoptotic activity against MCF-7 cells by upregulating the expression of p53, bax and casp3 and downregulating bcl2. Furthermore, NF-κB activity was abolished while the expression of TNfα was increased confirming the significant apoptotic effect of EGA-EML-APA. In conclusion, apamin-functionalized emulsomes have been successfully proposed as a potential anti-breast cancer formulation.

Cardiac small-conductance calcium-activated potassium channels in health and disease.

Small-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

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.

Natural products present neurotrophic properties in neurons of the limbic system in aging rodents.

Aging is a complex process that can lead to neurodegeneration and, consequently, several pathologies, including dementia. Physiological aging leads to changes in several body organs, including those of the central nervous system (CNS). Morphological changes in the CNS and particularly the brain result in motor and cognitive deficits affecting learning and memory and the circadian cycle. Characterizing neural modifications is critical to designing new therapies to target aging and associated pathologies. In this review, we compared aging to the changes occurring within the brain and particularly the limbic system. Then, we focused on key natural compounds, apamin, cerebrolysin, Curcuma longa, resveratrol, and N-PEP-12, which have shown neurotrophic effects particularly in the limbic system. Finally, we drew our conclusions delineating future perspectives for the development of novel natural therapeutics to ameliorate aging-related processes.

Apamin administration impact on miR-219 and miR-155-3p expression in cuprizone induced multiple sclerosis model.

Multiple sclerosis (MS) is a chronic debilitating disease that attacks the central nervous system. This study aims to investigate miR-219 and miR-155-3p expression levels involved in the myelination process following the administration of apamin peptide in the model of multiple sclerosis disease. Forty-four 8 week C57BL/6 male mice (22 ± 5 g) randomly divided into six groups. Apamin (100 µg/kg/BW) was administered intraperitoneally as a co-treatment during phase I (demyelination) or post-treatment phase II (remyelination) twice a week in cuprizone induced MS model. At the end of study myelin content and microRNA expression levels were measured with LFB staining and quantitative Real-Time PCR method, respectively. It was observed that the intended microRNAs were dysregulated during the different phases of disease induction. After 6 weeks of cuprizone exposure, miR-219 downregulated in phase I in comparison with the negative control. On the other hand, the apamin co-treatment significantly inhibit the miR-155-3p upregulation during the phase I as compared with the cuprizone group (p < 0.0001). Apamin has more impact on the miR155-3p reduction in phase I than miR-219 elevation in phase II. It could be considered as a therapeutic option for decreasing plaque formation during the exacerbation phase of the MS disease. Apamin has more impact on the miR155-3p reduction in phase I than miR-219 elevation in phase II. It could be considered as a therapeutic option for decreasing plaque formation during the exacerbation phase of the MS disease.

Apamin Suppresses LPS-Induced Neuroinflammatory Responses by Regulating SK Channels and TLR4-Mediated Signaling Pathways.

Neuroinflammation plays a vital role in neurodegenerative conditions. Microglia are a key component of the neuroinflammatory response. There is a growing interest in developing drugs to target microglia and thereby control neuroinflammatory processes. Apamin (APM) is a specifically selective antagonist of small conductance calcium-activated potassium (SK) channels. However, its effect on neuroinflammation is largely unknown. We examine the effects of APM on lipopolysaccharide (LPS)-stimulated BV2 and rat primary microglial cells. Regarding the molecular mechanism by which APM significantly inhibits proinflammatory cytokine production and microglial cell activation, we found that APM does so by reducing the expression of phosphorylated CaMKII and toll-like receptor (TLR4). In particular, APM potently suppressed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/signal transducer and activator of transcription (STAT)3 and phosphorylated mitogen-activated protein kinases (MAPK)-extracellular signal-regulated kinase (ERK). In addition, the correlation of NF-κB/STAT3 and MAPK-ERK in the neuroinflammatory response was verified through inhibitors. The literature and our findings suggest that APM is a promising candidate for an anti-neuroinflammatory agent and can potentially be used for the prevention and treatment of various neurological disorders.

Antioxidative, Antiapoptotic, and Anti-Inflammatory Effects of Apamin in a Murine Model of Lipopolysaccharide-Induced Acute Kidney Injury.

Sepsis is the major cause of acute kidney injury (AKI) in severely ill patients, but only limited therapeutic options are available. During sepsis, lipopolysaccharide (LPS), an endotoxin derived from bacteria, activates signaling cascades involved in inflammatory responses and tissue injury. Apamin is a component of bee venom and has been shown to exert antioxidative, antiapoptotic, and anti-inflammatory activities. However, the effect of apamin on LPS-induced AKI has not been elucidated. Here, we show that apamin treatment significantly ameliorated renal dysfunction and histological injury, especially tubular injury, in LPS-injected mice. Apamin also suppressed LPS-induced oxidative stress through modulating the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and heme oxygenase-1. Moreover, tubular cell apoptosis with caspase-3 activation in LPS-injected mice was significantly attenuated by apamin. Apamin also inhibited cytokine production and immune cell accumulation, suppressed toll-like receptor 4 pathway, and downregulated vascular adhesion molecules. Taken together, these results suggest that apamin ameliorates LPS-induced renal injury through inhibiting oxidative stress, apoptosis of tubular epithelial cells, and inflammation. Apamin might be a potential therapeutic option for septic AKI.

Expanding the MiniAp-4 BBB-shuttle family: Evaluation of proline cis-trans ratio as tool to fine-tune transport.

Venoms have recently emerged as a promising field in drug discovery due to their good selectivity and affinity for a wide range of biological targets. Among their multiple potential applications, venoms are a rich source of blood-brain barrier (BBB) peptide shuttles. We previously described a short nontoxic derivative of apamin, MiniAp-4, which can transport a wide range of cargoes across the BBB. Here, we have studied the conformation of the proline residue of a range of MiniAp-4 analogues by high-field NMR techniques, with the aim to identify whether there is a direct relation between the cis/trans population and a range of features, such as the capacity to transport molecules across a human-based cellular model and stability in various media. The most promising candidate showed improved transport properties for a relevant small fluorophore.

Calcium-activated SK potassium channels are key modulators of the pacemaker frequency in locus coeruleus neurons.

The physiological, intrinsic activity of noradrenergic locus coeruleus (LC) neurons is important for the control of sleep/wakefulness, cognition and autonomous body functions. Dysregulations of the LC-noradrenergic network contribute to the pathogenesis of psychiatric disorders and are key findings in early stages of neurodegenerative diseases. Therefore, identifying ion channels mediating the intrinsic pacemaking mechanism of LC neurons, which is in turn directly coupled to Ca2+ homeostasis and cell survival signaling pathways, can help to foster our understanding of the vulnerability of these neurons in neurodegenerative diseases. Small-conductance Ca2+-activated K+ (SK) channels regulate the intrinsic firing patterns in different central neurons and are essential regulators of the intracellular Ca2+ homeostasis. However, the role of SK channels for the intrinsic pacemaking of LC neurons in mice is still unclear. Therefore we performed qPCR expression analysis as well as patch clamp recordings of in vitro brainstem slices, for instance testing SK channel blockers and activators like apamin and NS309, respectively. Although we found a transcriptional expression of SK1, SK2 and SK3 channels, SK2 was the predominantly expressed subunit in mouse LC neurons. Using perforated-patch clamp experiments, we found that SK channels are essential regulators of the intrinsic pacemaking of LC neurons, mediating a large fraction of the afterhyperpolarization (AHP) in these cells. Consistent with a previous observation that a concerted action of L- and T-type Cav channels is essential for the pacemaking of LC neurons, we found that SK channel activation, and the respective AHP amplitude, is primarily coupled to Ca2+ influx via these types of Ca2+ channels. Our study identified SK2 channels as drug targets for the tuning of the pacemaker frequency in disorders involving a dysregulation of the LC.

The small neurotoxin apamin blocks not only small conductance Ca2+ activated K+ channels (SK type) but also the voltage dependent Kv1.3 channel.

Apamin is frequently used as a specific blocker of small-conductance Ca2+-activated (SK type) K+ channels. Here we show that the small neurotoxin is not as specific as anticipated. It is also a high-affinity inhibitor with an IC50 of 13 nM of the Kv1.3 channel; it blocks the latter with potency similar to the Kv1.3 blocker PAP-1. Since SK type channels and Kv1.3 channels are frequently coexpressed in different tissues such as cells of the immune system, apamin must be used with caution as a pharmacological tool.

MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery.

Drug delivery across the blood-brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non-invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam-bridged peptidomimetic MiniAp-4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human-cell-based BBB model. Furthermore, MiniAp-4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.

Coassembly and coupling of SK2 channels and mGlu5 receptors.

Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca(2+) wave-dependent activation of small-conductance Ca(2+)-activated K(+) (SK) channels. Here, we show that mGlu5 receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5 receptor stimulation activated apamin-sensitive SK2-mediated K(+) currents. In addition, coexpression of mGlu5 receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5 receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5 receptors and SK2 channels that reflects their molecular coassembly.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

Apamin-Sensitive K+ Current Upregulation in Volume-Overload Heart Failure is Associated with the Decreased Interaction of CK2 with SK2.

Recent studies have shown that the sensitivity of apamin-sensitive K(+) current (I KAS, mediated by apamin-sensitive small conductance calcium-activated potassium channels subunits) to intracellular Ca(2+) is increased in heart failure (HF), leading to I KAS upregulation, action potential duration shortening, early after depolarization, and recurrent spontaneous ventricular fibrillation. We hypothesized that casein kinase 2 (CK2) interacted with small conductance calcium-activated potassium channels (SK) is decreased in HF, and protein phosphatase 2A (PP2A) is increased on the opposite, upregulating the sensitivity of I KAS to intracellular Ca(2+) in HF. Rat model of volume-overload HF was established by an abdominal arteriovenous fistula procedure. The expression of SK channels, PP2A and CK2 was detected by Western blot analysis. Interaction and colocalization of CK2 with SK channel were detected by co-immunoprecipitation analysis and double immunofluorescence staining. In HF rat left ventricle, SK3 was increased by 100 % (P < 0.05), and SK2 was not significantly changed. PP2A protein was increased by 94.7 % in HF rats (P < 0.05), whereas the level of CK2 was almost unchanged. We found that CK2 colocalized with SK2 and SK3 in rat left ventricle. With anti-CK2α antibody, SK2 and SK3 were immunoprecipitated, the level of precipitated SK2 decreased by half, whereas precipitated SK3 was almost unchanged. In conclusion, the increased expression of total PP2A and decreased interaction of CK2 with SK2 may underlie enhanced sensitivity of I KAS to intracellular Ca(2+) in volume-overload HF rat.