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Background: Hypoxic pulmonary vascular remodeling (HPVR) is a key pathological feature of hypoxic pulmonary hypertension (HPH). Oxygen-sensitive potassium (K + ) channels in pulmonary artery smooth muscle cells (PASMCs) play a crucial role in HPVR. Luteolin (Lut) is a plant-derived flavonoid compound with variety of pharmacological actions. Our previous study found Lut alleviated HPVR in HPH rat., Purpose: To elucidate the mechanism by which Lut mitigated HPVR, focusing on oxygen-sensitive voltage-dependent potassium channel 1.5 (Kv1.5)., Methods: HPH rat model was established using hypobaric chamber to simulate 5000 m altitude. Isolated perfused/ventilated rat lung, isolated pulmonary arteriole ring was utilized to investigate the impact of Lut on K + channels activity. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was assessed. CyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-3 levels in lung tissue of HPH rat were tested. The effect of Lut on Kv1.5, cytoplasmic free calcium concentration ([Ca 2+ ] cyt ), CyclinD1, CDK4, PCNA, Bax/Bcl-2 was examined in PASMCs under hypoxia, with DPO-1 as a Kv1.5 specific inhibitor. The binding affinity between Lut and Kv1.5 in PASMCs was detected by drug affinity responsive target stability (DARTS). The overexpression of KCNA5 gene (encoding Kv1.5) in HEK293T cells was utilized to confirm the interaction between Lut and Kv1.5. Furthermore, the impact of Lut on mitochondrial structure, SOD, GSH, GSH-Px, MDA and HIF-1α levels were evaluated in lung tissue of HPH rat and PASMCs under hypoxia., Results: Lut dilated pulmonary artery by directly activating Kv and Ca 2+ -activated K + channels (K Ca ) in smooth muscle. Kv1.5 level in lung tissue and pulmonary arteriole of HPH rat was upregulated by Lut. Lut downregulated CyclinD1, CDK4, PCNA while upregulating Bax/Bcl-2/caspase-3 axis in lung tissue of HPH rat. Lut decreased [Ca 2+ ] cyt , reduced CDK4, CyclinD1, PCNA, increased Bax/Bcl-2 ratio, in PASMCs under hypoxia, by upregulating Kv1.5. The binding affinity and the interaction between Lut and Kv1.5 was verified in PASMCs and in HEK293T cells. Lut also decreased [Ca 2+ ] cyt and inhibited proliferation via targeting Kv1.5 of HEK293T cells under hypoxia. Furthermore, Lut protected mitochondrial structure, increased SOD, GSH, GSH-Px, decreased MDA, in lung tissue of HPH rat. Lut downregulated HIF-1α level in both lung tissue of HPH rat and PASMCs under hypoxia., Conclusion: Lut alleviated HPVR by promoting vasodilation of pulmonary artery, reducing cellular proliferation, and inducing apoptosis through upregulating of Kv1.5 in PASMCs., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K + current (I Kur ) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5 ) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven KCNA5 variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. KCNA5 variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1%. The data indicate that some KCNA5 variants found in patients with PAH have critical consequences for channel function, supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

The voltage-gated K + channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K + current (I Kur ) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H 2 S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H 2 S producing enzymes protein expression in HL-1 cardiomyocytes. H 2 S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H 2 S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of I kur by H 2 S has important cardiovascular implications and represents a novel and potential therapeutic target., (© 2023. The Author(s).)

In this paper, the influence of external terahertz electromagnetic fields with different frequencies of 4 THz, 10 THz, 15 THz, and 20 THz on the permeability of the Kv1.2 voltage-gated potassium ion channel on the nerve cell membrane was studied using the combined model of the "Constant Electric Field-Ion Imbalance" method by molecular dynamics. We found that although the applied terahertz electric field does not produce strong resonance with the -C=O groups of the conservative sequence T-V-G-Y-G amino acid residue of the selective filter (SF) of the channel, it would affect the stability of the electrostatic bond between potassium ions and the carbonyl group of T-V-G-Y-G of SF, and it would affect the stability of the hydrogen bond between water molecules and oxygen atoms of the hydroxyl group of the 374THR side chain at the SF entrance, changing the potential and occupied states of ions in the SF and the occurrence probability of the permeation mode of ions and resulting in the change in the permeability of the channel. Compared with no external electric field, when the external electric field with 15 THz frequency is applied, the lifetime of the hydrogen bond is reduced by 29%, the probability of the "soft knock on" mode is decreased by 46.9%, and the ion flux of the channel is activated by 67.7%. Our research results support the view that compared to "direct knock-on", "soft knock-on" is a slower permeation mode.

Kv1.5 potassium channel, encoded by KCNA5, is a promising target for the treatment of atrial fibrillation, one of the common arrhythmia. A new series of arylmethylpiperidines derivatives based on DDO-02001 were synthesised and evaluated for their ability to inhibit Kv1.5 channel. Among them, compound DDO-02005 showed good inhibitory activity (IC 50 = 0.72 μM), preferable anti-arrhythmic effects and favoured safety. These results indicate that DDO-02005 can be a promising Kv1.5 inhibitor for further studies.

Among voltage-gated potassium channel (K V ) isoforms, K V 1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of K V 1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to K V 1.6. Previously, we have demonstrated that K V isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of K V 1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC 50 of ≈70 nM against K V 1.3. Quite surprisingly, Tk-hefu-11 turns out to block K V 1.6 with even higher potency, presenting an EC 50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and K V channels to explain the dramatic increase in K V 1.6 affinity. Since K V 1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of K V 1.6 and its functions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1., Methods and Results: Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1β secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1β. MSU reduced intracellular K + concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1β, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1β and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5., Conclusions: Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF., (© 2022. The Author(s).)

Both inherited and acquired cardiac arrhythmias are often associated with the abnormal functional expression of ion channels at the cellular level. The complex machinery that continuously traffics, anchors, organizes, and recycles ion channels at the plasma membrane of a cardiomyocyte appears to be a major source of channel dysfunction during cardiac arrhythmias. This has been well established with the discovery of mutations in the genes encoding several ion channels and ion channel partners during inherited cardiac arrhythmias. Fibrosis, altered myocyte contacts, and post-transcriptional protein changes are common factors that disorganize normal channel trafficking during acquired cardiac arrhythmias. Channel availability, described notably for hERG and K V 1.5 channels, could be another potent arrhythmogenic mechanism. From this molecular knowledge on cardiac arrhythmias will emerge novel antiarrhythmic strategies.

Voltage-dependent K + (Kv) channels play the role of returning the membrane potential to the resting state, thereby maintaining the vascular tone. Here, we used native smooth-muscle cells from rabbit coronary arteries to investigate the inhibitory effect of lorcainide, a class Ic antiarrhythmic agent, on Kv channels. Lorcainide inhibited Kv channels in a concentration-dependent manner with an IC 50 of 4.46 ± 0.15 μM and a Hill coefficient of 0.95 ± 0.01. Although application of lorcainide did not change the activation curve, it shifted the inactivation curve toward a more negative potential, implying that lorcainide inhibits Kv channels by changing the channels' voltage sensors. The recovery time constant from channel inactivation increased in the presence of lorcainide. Furthermore, application of train steps (of 1 or 2 Hz) in the presence of lorcainide progressively augmented the inhibition of Kv currents, implying that lorcainide-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with Kv1.5 or Kv2.1/2.2 inhibitors effectively reduced the amplitude of the Kv current but did not affect the inhibitory effect of lorcainide. Based on these results, we conclude that lorcainide inhibits vascular Kv channels in a concentration and use (state)-dependent manner by changing their inactivation gating properties. Considering the clinical efficacy of lorcainide, and the pathophysiological significance of vascular Kv channels, our findings should be considered when prescribing lorcainide to patients with arrhythmia and vascular disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the 'NACHT, LRR, and PYD Domains-containing Protein 3' (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF., Methods and Results: Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3-/-) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3-/- mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K+-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca2+ release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3-/-) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca2+ release in DIO mice., Conclusion: These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

The atrial-specific ultra-rapid delayed rectifier K + current (Ikur) plays an important role in the progression of atrial fibrillation (AF). Because inflammation is known to lead to the onset of AF, we aimed to investigate whether tumour necrosis factor-α (TNF-α) played a role in regulating Ikur and the potential signalling pathways involved. Whole-cell patch-clamp and biochemical assays were used to study the regulation and expression of Ikur in myocytes and in tissues from left atrial appendages (LAAs) obtained from patients with sinus rhythm (SR) or AF, as well as in rat cardiomyocytes (H9c2 cells) and mouse atrial myocytes (HL-1 cells). Ikur current density was markedly reduced in atrial myocytes from AF patients compared with SR controls. Reduction of Kv1.5 protein levels was accompanied by increased expression of TNF-α and protein kinase C (PKC)α activation in AF patients. Treatment with TNF-α dose-dependently reduced Ikur and protein expression of Kv1.5 but not Kv3.1b in H9c2 cells and HL-1 cells. TNF-α also increased activity of PKCα. Specific PKCα inhibitor Gö6976 alleviated the reduction in Ikur induced by TNF-α, but not the reduction in Kv1.5 protein. TNF-α was involved in the electrical remodelling associated with AF, probably by depressing Ikur in atrial myocytes via activation of PKCα., (© 2020 John Wiley & Sons Australia, Ltd.)

(1) Background: As membrane channels contribute to different cell functions, understanding the underlying mechanisms becomes extremely important. A large number of neuronal channels have been investigated, however, less studied are the channels expressed in the glia population, particularly in microglia. In the present study, we focused on the function of the Kv1.3, Kv1.5 and Kir2.1 potassium channels expressed in both BV2 cells and primary microglia cultures, which may impact the cellular migration process. (2) Methods: Using an immunocytochemical approach, we were able to show the presence of the investigated channels in BV2 microglial cells, record their currents using a patch clamp and their role in cell migration using the scratch assay. The migration of the primary microglial cells in culture was assessed using cell culture inserts. (3) Results: By blocking each potassium channel, we showed that Kv1.3 and Kir2.1 but not Kv1.5 are essential for BV2 cell migration. Further, primary microglial cultures were obtained from a line of transgenic CX3CR1-eGFP mice that express fluorescent labeled microglia. The mice were subjected to a spared nerve injury model of pain and we found that microglia motility in an 8 µm insert was reduced 2 days after spared nerve injury (SNI) compared with sham conditions. Additional investigations showed a further impact on cell motility by specifically blocking Kv1.3 and Kir2.1 but not Kv1.5; (4) Conclusions: Our study highlights the importance of the Kv1.3 and Kir2.1 but not Kv1.5 potassium channels on microglia migration both in BV2 and primary cell cultures.

Abnormal proliferation of granulosa cells is implicated in ovarian dysfunction and dysregulated folliculogenesis in the polycystic ovary syndrome (PCOS). Aberrant microRNA (miRNA) expression might contribute to disordered folliculogenesis and granulosa cell proliferation in PCOS. This study aimed to investigate the roles of miR-3188 in ovarian dysfunction, as well as the mechanism involved in granulosa cell proliferation in PCOS. Firstly, peripheral blood samples were isolated from PCOS patients and healthy controls, and qRT-PCR analysis demonstrated a dramatic increase in miR-3188 in PCOS patients when compared to the healthy controls. Secondly, miR-3188 overexpression increased cell viability of the granulosa-like tumor cell line (KGN). However, cell viability of KGN was repressed by interference with miR-3188. MiR-3188 promoted cell cycle of KGN through increasing cyclinD1 and decreasing p21 levels. Moreover, cell apoptosis was suppressed by miR-3188 in KGN, indicated by enhanced Bcl-2, and reduced Bax and cleaved caspase-3 levels, whereas knockdown of miR-3188 resulted in opposite effects. Lastly, potassium voltage-gated channel subfamily A member 5 (KCNA5) was verified as a target of miR-3188. KCNA5 expression was decreased and displayed negative correlation with miR-3188 levels in PCOS patients. Overexpression of KCNA5 attenuated the promotive effects of miR-3188 on cell viability and cell cycle in KGN. In conclusion, miR-3188, a key miRNA enhanced in PCOS, promoted granulosa cell proliferation through down-regulation of KCNA5, providing a new therapeutic target for PCOS.

K V 1.5 channel function is modified by different regulatory subunits. K V β1.3 subunits assemble with K V 1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the K V β1.3-induced fast inactivation, decreases the amplitude of the current K V 1.5-K V β1.3 and modifies their pharmacology likely due to changes in the traffic of K V 1.5-K V β1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with K V 1.5-K V β1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of K V 1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the K V 1.5-K V β1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of K V 1.5-K V β1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.

Darifenacin, an anticholinergic agent, has been used to treat overactive bladder syndrome. Despite its extensive clinical use, there is little information about the effect of darifenacin on vascular ion channels, specifically K + channels. This study aimed to investigate the effect of the anti-muscarinic drug darifenacin on voltage-gated K + (Kv) channels, vascular contractility, and coronary blood flow in rabbit coronary arteries. We used the whole-cell patch-clamp technique to evaluate the effect of darifenacin on Kv channels. Darifenacin inhibited the Kv current in a concentration-dependent manner. Applying 1 μM darifenacin shifted the activation and inactivation curves toward a more positive and negative potential, respectively. Darifenacin slowed the time constants of recovery from inactivation. Furthermore, blockade of the Kv current with darifenacin was increased gradually by applying a train of pulses, indicating that darifenacin inhibited Kv currents in a use- (state)-dependent manner. The darifenacin-mediated inhibition of Kv currents was associated with the Kv1.5 subtype, not the Kv2.1 or Kv7 subtype. Applying another anti-muscarinic drug atropine or ipratropium did not affect the Kv current or change the inhibitory effect of darifenacin. Isometric organ bath experiments using isolated coronary arteries were applied to evaluate whether darifenacin-induced inhibition of the Kv channel causes vasocontraction. Darifenacin substantially induced vasocontraction. Furthermore, darifenacin caused membrane depolarization and decreased coronary blood flow. From these results, we concluded that darifenacin inhibits the Kv currents in concentration- and use- (state)-dependent manners. Inhibition of the Kv current with darifenacin occurred by shifting the steady-state activation and inactivation curves regardless of its anti-muscarinic effect., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Polychlorinated biphenyls (PCBs) are persistent and serious organic pollutants and can theoretically form 209 congeners. PCBs can be divided into two categories: dioxin-like (DL) and non-DL (NDL). NDL-PCBs, which lack aryl hydrocarbon receptor affinity, have been shown to perturb the functions of Jurkat T cells, cerebellar granule cells, and uterine cells. Kv1.3 and Kv1.5 channels are important in immune and heart functions, respectively. We investigated the acute effects of 2,2',6-trichlorinated biphenyl (PCB19), an NDL-PCB, on the currents of human Kv1.3 and Kv1.5 channels. PCB19 acutely blocked the Kv1.3 peak currents concentration-dependently with an IC 50 of ~2 μM, without changing the steady-state current. The PCB19-induced inhibition of the Kv1.3 peak current occurred rapidly and voltage-independently, and the effect was irreversible, excluding the possibility of genomic regulation. PCB19 increased the time constants of both activation and inactivation of Kv1.3 channels, resulting in the slowing down of both ultra-rapid activation and intrinsic inactivation. However, PCB19 failed to alter the steady-state curves of activation and inactivation. Regarding the Kv1.5 channel, PCB19 affected neither the peak current nor the steady-state current at the same concentrations tested in the Kv1.3 experiments, showing selective inhibition of PCB19 on the Kv1.3 than the Kv1.5. The presented data indicate that PCB19 could acutely affect the human Kv1.3 channel through a non-genomic mechanism, possibly causing toxic effects on various human physiological functions related to the Kv1.3 channel, such as immune and neural systems., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (I Kv1.5 ) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Gastric and colorectal cancers are the most common malignant tumours, leading to a significant number of cancer-related deaths worldwide. Recently, increasing evidence has demonstrated that cancer cells exhibit a differential expression of potassium channels and this can contribute to cancer progression. However, their expression and localisation at the somatic level remains uncertain. In this study, we have investigated the expression levels of KCNB1 and KCNA5 genes encoding ubiquitous Kv2.1 and Kv1.5 potassium channels in gastric and colorectal tumours., Methods: Gastric and colorectal tumoral and peritumoral tissues were collected to evaluate the expression of KCNB1 and KCNA5 mRNA by quantitative PCR. Moreover, the immunohistochemical staining profile of Kv2.1 and Kv1.5 was assessed on 40 Formalin-Fixed and Paraffin-Embedded (FFPE) gastric carcinoma tissues. Differences in gene expression between tumoral and peritumoral tissues were compared statistically with the Mann-Whitney U test. The association between the clinicopathological features of the GC patients and the expression of both Kv proteins was investigated with χ2 and Fisher's exact tests., Results: The mRNA fold expression of KCNB1 and KCNA5 genes showed a lower mean in the tumoral tissues (0.06 ± 0.17, 0.006 ± 0.009) compared to peritumoral tissues (0.08 ± 0.16, 0.16 ± 0.48, respectively) without reaching the significance rate (p = 0.861, p = 0.152, respectively). Interestingly, Kv2.1 and Kv1.5 immunostaining was detectable and characterised by a large distribution in peritumoral and tumoral epithelial cells. More interestingly, inflammatory cells were also stained. Surprisingly, Kv2.1 and Kv1.5 staining was undoubtedly and predominantly detected in the cytoplasm compartment of tumour cells. Indeed, the expression of Kv2.1 in tumour cells revealed a significant association with the early gastric cancer clinical stage (p = 0.026)., Conclusion: The data highlight, for the first time, the potential role of Kv1.5 and Kv2.1 in gastrointestinal-related cancers and suggests they may be promising prognostic markers for these tumours.

The opening and closing of voltage-gated ion channels are regulated by voltage sensors coupled to a gate that controls the ion flux across the cellular membrane. Modulation of any part of gating constitutes an entry point for pharmacologically regulating channel function. Here, we report on the discovery of a large family of warfarin-like compounds that open the two voltage-gated type 1 potassium (K V 1) channels K V 1.5 and Shaker, but not the related K V 2-, K V 4-, or K V 7-type channels. These negatively charged compounds bind in the open state to positively charged arginines and lysines between the intracellular ends of the voltage-sensor domains and the pore domain. This mechanism of action resembles that of endogenous channel-opening lipids and opens up an avenue for the development of ion-channel modulators., Competing Interests: The authors declare no competing interest.

The modulation of voltage-gated K + (Kv) channels, involved in cell proliferation, arises as a potential therapeutic approach for the prevention of intimal hyperplasia present in in-stent restenosis (ISR) and allograft vasculopathy (AV). We studied the effect of PAP-1, a selective blocker of Kv1.3 channels, on development of intimal hyperplasia in vitro and in vivo in 2 porcine models of vascular injury. In vitro phenotypic modulation of VSMCs was associated to an increased functional expression of Kv1.3 channels, and only selective Kv1.3 channel blockers were able to inhibit porcine VSMC proliferation. The therapeutic potential of PAP-1 was then evaluated in vivo in swine models of ISR and AV. At 15-days follow-up, morphometric analysis demonstrated a substantial reduction of luminal stenosis in the allografts treated with PAP-1 (autograft 2.72 ± 1.79 vs allograft 10.32 ± 1.92 vs allograft + polymer 13.54 ± 8.59 vs allograft + polymer + PAP-1 3.06 ± 1.08 % of luminal stenosis; P = 0.006) in the swine model of femoral artery transplant. In the pig model of coronary ISR, using a prototype of PAP-1-eluting stent, no differences were observed regarding % of stenosis compared to control stents (31 ± 13 % vs 37 ± 18%, respectively; P = 0.372) at 28-days follow-up. PAP-1 treatment was safe and did not impair vascular healing in terms of delayed endothelialization, inflammation or thrombosis. However, an incomplete release of PAP-1 from stents was documented. We conclude that the use of selective Kv1.3 blockers represents a promising therapeutic approach for the prevention of intimal hyperplasia in AV, although further studies to improve their delivery method are needed to elucidate its potential in ISR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a rare and severe cardiopulmonary disease without curative treatments. PAH is a multifactorial disease that involves genetic predisposition, epigenetic factors, and environmental factors (drugs, toxins, viruses, hypoxia, and inflammation), which contribute to the initiation or development of irreversible remodeling of the pulmonary vessels. The recent identification of loss-of-function mutations in KCNK3 (KCNK3 or TASK-1) and ABCC8 (SUR1), or gain-of-function mutations in ABCC9 (SUR2), as well as polymorphisms in KCNA5 (Kv1.5), which encode two potassium (K + ) channels and two K + channel regulatory subunits, has revived the interest of ion channels in PAH. This review focuses on KCNK3, SUR1, SUR2, and Kv1.5 channels in pulmonary vasculature and discusses their pathophysiological contribution to and therapeutic potential in PAH.

Background: Cardiac gene expression and arrhythmia occurrence have time-of-day variation; however, daily changes in cardiac electrophysiology, arrhythmia susceptibility, and Ca 2+ handling have not been characterized. Furthermore, how these patterns change with age is unknown., Methods: Hearts were isolated during the light (zeitgeber time [ZT] 4 and ZT9) and dark cycle (ZT14 and ZT21) from adult (12-18 weeks) male mice. Hearts from aged (18-20 months) male mice were isolated at ZT4 and ZT14. All hearts were Langendorff-perfused for optical mapping with voltage- and Ca 2+ -sensitive dyes (n=4-7/group). Cardiac gene and protein expression were assessed with real-time polymerase chain reaction (n=4-6/group) and Western blot (n=3-4/group)., Results: Adult hearts had the shortest action potential duration (APD) and Ca 2+ transient duration (CaTD) at ZT14 (APD 80 : ZT4: 45.4±4.1 ms; ZT9: 45.1±8.6 ms; ZT14: 34.7±4.2 ms; ZT21: 49.2±7.6 ms, P <0.05 versus ZT4 and ZT21; and CaTD 80 : ZT4: 70.1±3.3 ms; ZT9: 72.7±2.7 ms; ZT14: 64.3±3.3 ms; ZT21: 74.4±1.2 ms, P <0.05 versus other time points). The pacing frequency at which CaT alternans emerged was faster, and average CaT alternans magnitude was significantly reduced at ZT14 compared with the other time points. There was a trend for decreased spontaneous premature ventricular complexes and pacing-induced ventricular arrhythmias at ZT14, and the hearts at ZT14 had diminished responses to isoproterenol compared with ZT4 (ZT4: 49.5.0±5.6% versus ZT14: 22.7±9.5% decrease in APD, P <0.01). In contrast, aged hearts exhibited no difference between ZT14 and ZT4 in nearly every parameter assessed (except APD 80 : ZT4: 39.7±1.9 ms versus ZT14: 33.8±3.1 ms, P <0.01). Gene expression of KCNA5 (potassium voltage-gated channel subfamily A member 5; encoding Kv1.5) was increased, whereas gene expression of ADRB1 (encoding β1-adrenergic receptors) was decreased at ZT14 versus ZT4 in adult hearts. No time-of-day changes in expression or phosphorylation of Ca 2+ handling proteins (SERCA2 [sarco/endoplasmic reticulum Ca 2+ -ATPase], RyR2 [ryanodine receptor 2], and PLB [phospholamban]) was found in ex vivo perfused adult isolated hearts., Conclusions: Isolated adult hearts have strong time-of-day variation in cardiac electrophysiology, Ca 2+ handling, and adrenergic responsiveness, which is disrupted with age.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K + channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K + channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K + channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC 50 = 2.7 and 24 μM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Background: Epidemiological studies have established obesity as an independent risk factor for atrial fibrillation (AF), but the underlying pathophysiological mechanisms remain unclear. Reduced cardiac sodium channel expression is a known causal mechanism in AF. We hypothesized that obesity decreases Nav1.5 expression via enhanced oxidative stress, thus reducing I Na , and enhancing susceptibility to AF., Methods: To elucidate the underlying electrophysiological mechanisms a diet-induced obese mouse model was used. Weight, blood pressure, glucose, F 2 -isoprostanes, NOX2 (NADPH oxidase 2), and PKC (protein kinase C) were measured in obese mice and compared with lean controls. Invasive electrophysiological, immunohistochemistry, Western blotting, and patch clamping of membrane potentials was performed to evaluate the molecular and electrophysiological phenotype of atrial myocytes., Results: Pacing-induced AF in 100% of diet-induced obese mice versus 25% in controls ( P <0.01) with increased AF burden. Cardiac sodium channel expression, I Na and atrial action potential duration were reduced and potassium channel expression (Kv1.5) and current ( I Kur ) and F 2 -isoprostanes, NOX2, and PKC-α/δ expression and atrial fibrosis were significantly increased in diet-induced obese mice as compared with controls. A mitochondrial antioxidant reduced AF burden, restored I Na , I Ca,L , I Kur , action potential duration, and reversed atrial fibrosis in diet-induced obese mice as compared with controls., Conclusions: Inducible AF in obese mice is mediated, in part, by a combined effect of sodium, potassium, and calcium channel remodeling and atrial fibrosis. Mitochondrial antioxidant therapy abrogated the ion channel and structural remodeling and reversed the obesity-induced AF burden. Our findings have important implications for the management of obesity-mediated AF in patients. Graphic Abstract: A graphic abstract is available for this article.

Background: High blood glucose impairs voltage-gated K + (Kv) channel-mediated vasodilation in rat coronary artery smooth muscle cells (CSMCs) via oxidative stress. Advanced glycation end product (AGE) and receptor for AGE (RAGE) axis has been found to impair coronary dilation by reducing Kv channel activity in diabetic rat small coronary arteries (RSCAs). However, its underlying mechanism remain unclear. Here, we used isolated arteries and primary CSMCs to investigate the effect of AGE incubation on Kv channel-mediated coronary dilation and the possible involvement of peroxisome proliferators-activated receptor (PPAR) -γ pathway., Methods: The RSCAs and primary CSMCs were isolated, cultured, and treated with bovine serum albumin (BSA), AGE-BSA, alagrebrium (ALA, AGE cross-linking breaker), pioglitazone (PIO, PPAR-γ activator) and/or GW9662 (PPAR-γ inhibitor). The groups were accordingly divided as control, BSA, AGE, AGE + ALA, AGE + PIO, or AGE + PIO + GW9662. Kv channel-mediated dilation was analyzed using wire myograph. Histology and immunohistochemistry of RSCAs were performed. Western blot was used to detect the protein expression of RAGE, major Kv channel subunits expressed in CSMCs (Kv1.2 and Kv1.5), PPAR-γ, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2)., Results: AGE markedly reduced Forskolin-induced Kv channel-mediated dilation of RSCAs by engaging with RAGE, and ALA or PIO significantly reversed the functional loss of Kv channel. In both RSCAs and CSMCs, AGE reduced Kv1.2/1.5 expression, increased RAGE and NOX-2 expression, and inhibited PPAR-γ expression, while ALA or PIO treatment partially reversed the inhibiting effects of AGE on Kv1.2/1.5 expression, accompanied by the downregulation of RAGE and decreased oxidative stress. Meanwhile, silencing of RAGE with siRNA remarkably alleviated the AGE-induced downregulation of Kv1.2/1.5 expression in CSMCs., Conclusion: AGE reduces the Kv channel expression in CSMCs and further impairs the Kv channel-mediated dilation in RSCAs. The AGE/RAGE axis may enhance oxidative stress by inhibiting the downstream PPAR-γ pathway, thus playing a critical role in the dysfunction of Kv channels.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific K V 1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current I Kur . Besides, regulated K V 1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of K V 1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of K V 1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile K V 1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static K V 1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial K V 1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok K V 1.5 channels in the plasma membrane., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Atrial fibrillation (AF), the most common sustained arrhythmia, significantly increases cardiovascular and cerebrovascular morbidity and mortality. The pathogenesis and treatment of AF remain a major challenge in the field of cardiology. We previously found that cold-inducible RNA-binding protein (CIRP) regulated ventricular repolarization by posttranscriptionally regulating Kv4.2/4.3 ion channels in rats, but the role of CIRP in AF is not clear., Objective: The purpose of this study was to confirm that CIRP participates in atrial electrophysiological remodeling and AF occurrence by regulating atrial channels posttranscriptionally., Methods: Programmed intra-atrial stimulation was used to induce AF in wild-type or transcription activator-like effector nucleases-based CIRP knockout (KO) rats. Atrial optical mapping, patch clamp, Western blotting, RNA immunoprecipitation, and luciferase reporter assays were performed to evaluate the underlying mechanism of atrial electrical remodeling., Results: First, we observed a shortened atrial effective refractory period and increased susceptibility to AF in CIRP KO rats. Second, atria-specific CIRP delivery through an adeno-associated viral vector serotype 9 prolonged the atrial effective refractory period and attenuated AF development in CIRP KO rats. Third, we observed the shortened action potential duration and enhanced expression of Kv1.5 and Kv4.2/4.3 in KO rats. The transient outward current blocker 4-Aminopyridine and ultrarapid component of the delayed rectifier current blocker Diphenyl phosphine oxide-1 restored the shortened action potential duration in KO atria. Finally, we demonstrated that CIRP suppressed Kv1.5 and Kv4.2/4.3 expression by directly targeting their 3'-untranslated regions., Conclusion: CIRP plays a protective role in preventing AF onset through the posttranscriptional regulation of Kv1.5 and Kv4.2/4.3., (Copyright © 2020 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Increased granulosa cell (GC) proliferation may contribute to abnormal folliculogenesis in patients with polycystic ovary syndrome (PCOS), which affects approximately 10% reproductive aged women. However, the mechanisms underlying increased GC proliferation in PCOS remain incompletely understood. In this study, we identified miR-3940-5p as a hub miRNA in GC from PCOS using weighted gene co-expression network analysis (WGCNA), and real-time polymerase chain reaction (RT-PCR) analysis confirmed that miR-3940-5p was significantly increased in GC from PCOS. Enrichment analysis of predicted target genes of miR-3940-5p indicated potential roles of miR-3940-5p in follicular development and cell proliferation regulation. Consistently, functional study confirmed that miR-3940-5p overexpression promoted granulosa cell proliferation. Integrated analysis of mRNA expression profiling data and predicted target genes of miR-3940-5p identified potassium voltage-gated channel subfamily A member 5 (KCNA5) as a potential target of miR-3940-5p, and was validated by luciferase reporter assay. Finally, functional analysis suggested that miR-3940-5p promoted GC proliferation in a KCNA5 dependent manner. In conclusion, miR-3940-5p was a hub miRNA upregulated in GC from PCOS, and promoted GC proliferation by targeting KCNA5., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Atrial fibrillation (AF) is prevalent in diabetes mellitus (DM); however, the basis for this is unknown. This study investigated AF susceptibility and atrial electrophysiology in type 1 diabetic Akita mice using in vivo intracardiac electrophysiology, high-resolution optical mapping in atrial preparations, and patch clamping in isolated atrial myocytes. qPCR and western blotting were used to assess ion channel expression. Akita mice were highly susceptible to AF in association with increased P-wave duration and slowed atrial conduction velocity. In a second model of type 1 DM, mice treated with streptozotocin (STZ) showed a similar increase in susceptibility to AF. Chronic insulin treatment reduced susceptibility and duration of AF and shortened P-wave duration in Akita mice. Atrial action potential (AP) morphology was altered in Akita mice due to a reduction in upstroke velocity and increases in AP duration. In Akita mice, atrial Na + current (I Na ) and repolarizing K + current (I K ) carried by voltage gated K + (K v 1.5) channels were reduced. The reduction in I Na occurred in association with reduced expression of SCN5a and voltage gated Na + (Na V 1.5) channels as well as a shift in I Na activation kinetics. Insulin potently and selectively increased I Na in Akita mice without affecting I K Chronic insulin treatment increased I Na in association with increased expression of Na V 1.5. Acute insulin also increased I Na , although to a smaller extent, due to enhanced insulin signaling via phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Our study reveals a critical, selective role for insulin in regulating atrial I Na , which impacts susceptibility to AF in type 1 DM., Competing Interests: The authors declare no competing interest.

The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (I Kv1.5 ) in a normal, isotonic solution. We observed that mechanical stretch increased I Kv1.5 , and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced I Kv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits I Kv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in I Kv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in I Kv1.5 ., (© 2020 Milton et al.)

Pulmonary hypertension (PH) is distal pulmonary arterial remodelling and is mainly due to the abnormal proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Apigenin, a natural dietary flavonoid, is a promising PH preventive agent that inhibits PASMC proliferation and induces apoptosis. In this study, we investigated the biological effects of apigenin on PH. PH was induced in male Sprague-Dawley rats by chronic hypoxia exposure. Administration of apigenin prevented the development of PH, hypoxia-induced right ventricular hypertrophy and pulmonary arterial remodelling and prevented the progression of established PH in this model. Moreover, treatment with apigenin induced mitochondria-dependent apoptosis. To explore the underlying mechanisms, the mitochondrial membrane potential (Δψm) and the mitochondria-dependent apoptosis factors cytochrome C, BAX, Bcl-2, cleaved caspase 3, and cleaved caspase 9 were analysed. These results confirmed that apigenin induces mitochondria-dependent apoptosis in hypoxic PASMCs to protect against PH. In addition, treatment with apigenin reversed hypoxia-induced inhibition of KV1.5 expression both in vivo and in vitro. The KV1.5 inhibitor diphenyl phosphine oxide-1 (DPO-1) abrogated apigenin-induced mitochondria-dependent apoptosis in hypoxic PASMCs, suggesting that KV1.5 is implicated in apigenin-induced mitochondria-dependent apoptosis. Furthermore, functional studies revealed that apigenin activated mitochondria-dependent apoptosis by modulation of hypoxia-induced factor 1α (HIF-1α) signalling. Together, our study shows that apigenin attenuates PH via inhibiting the HIF-1α-KV1.5 channel pathway to induce PASMC mitochondria-dependent apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The voltage-gated potassium channel Kv1.5, which mediates the cardiac ultra-rapid delayed-rectifier (I Kur ) current in human cells, has a crucial role in atrial fibrillation. Therefore, the design of selective Kv1.5 modulators is essential for the treatment of pathophysiological conditions involving Kv1.5 activity. This review summarizes the progress of molecular structures and the functionality of different types of Kv1.5 modulators, with a focus on clinical cardiovascular drugs and a number of active natural products, through a summarization of 96 compounds currently widely used. Furthermore, we also discuss the contributions of Kv1.5 and the regulation of the structure-activity relationship (SAR) of synthetic Kv1.5 inhibitors in human pathophysiology. SAR analysis is regarded as a useful strategy in structural elucidation, as it relates to the characteristics that improve compounds targeting Kv1.5. Herein, we present previous studies regarding the structural, pharmacological, and SAR information of the Kv1.5 modulator, through which we can assist in identifying and designing potent and specific Kv1.5 inhibitors in the treatment of diseases involving Kv1.5 activity., Competing Interests: The authors declare no conflict of interest.

Our previous investigation indicated that angiotensin II (Ang II) enhances the expression of Kv1.5, a promising target for the treatment of atrial fibrillation (AF), by activating reactive oxygen species (ROS)-dependent phosphorylation of Smad 2/3 (forming P-Smad 2/3) and ERK 1/2 (forming P-ERK 1/2). A recent study indicated that aldosterone (Aldo) upregulates atrial Kv1.5 protein in a rat AF model, but the mechanism remains unknown. The present study aimed to clarify the mechanism underlying Aldo-induced Kv1.5 expression and to test whether spironolactone may modulate atrial Kv1.5. Our Western blot analysis indicated that the Aldo/mineralocorticoid receptor (MR) interacts with Ang II/AT 1 R in upregulating Kv1.5 expression in cultured neonatal atrial myocytes (NRAMs). Blockade of MR with spironolactone and of AT 1 R with losartan significantly suppressed Kv1.5 expression induction by combined Aldo and Ang II treatment. Aldo increased the protein expression of Nox1, Nox2 and Nox4, but this effect was abolished by spironolactone pretreatment. The Aldo-induced upregulation of Kv1.5 was also reversed by the Src protein tyrosine kinase family inhibitor PP2, the Nox2 inhibitor gp91ds-tat and the Nox1/Nox4 inhibitor GKT137831 but not by the Rac GTPase inhibitor NSC23766. Flow cytometry showed that the Aldo-induced ROS production was inhibited by spironolactone, PP2, gp91ds-tat and GKT137831. Spironolactone suppressed the Aldo-induced protein expression phosphorylated Src (P-Src), P-Smad 2/3 and P-ERK 1/2. In conclusion, we have demonstrated that spironolactone suppresses Aldo-induced Kv1.5 expression by attenuating MR-Nox1/2/4-mediated ROS generation in NRAMs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is a chronic, inflammatory, neurodegenerative disease with an autoimmune component. It was suggested that potassium channels, which are involved in crucial biological functions may have a role in different diseases, including MS and its animal model, experimental autoimmune encephalomyelitis (EAE). It was shown that voltage-gated potassium channels Kv1.5 are responsible for fine-tuning in the immune physiology and influence proliferation and differentiation in microglia and astrocytes. Here, we explored the cellular distribution of the Kv1.5 channel, together with its transcript and protein expression in the male rat spinal cord during different stages of EAE. Our results reveal a decrease of Kv1.5 transcript and protein level at the peak of disease, where massive infiltration of myeloid cells occurs, together with reactive astrogliosis and demyelination. Also, we revealed that the presence of this channel is not found in infiltrating macrophages/microglia during EAE. It is interesting to note that Kv1.5 channel is expressed only in resting microglia in the naïve animals. Predominant expression of Kv1.5 channel was found in the astrocytes in all experimental groups, while some vimentin + cells, resembling macrophages, are devoid of Kv1.5 expression. Our results point to the possible link between Kv1.5 channel and the pathophysiological processes in EAE.

A pair of stereoisomers possessing novel structures with 6/6/5 fused-ring systems, neo-debromoaplysiatoxin E ( 1 ) and neo-debromoaplysiatoxin F ( 2 ), were isolated from the marine cyanobacterium Lyngbya sp. Their structures were elucidated using various spectroscopic techniques including high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR). The absolute stereochemistry was determined by calculated electronic circular dichroism (ECD) and gauge-independent atomic orbital (GIAO) NMR shift calculation followed by DP4+ analysis. Significantly, this is the first report on aplysiatoxin derivatives with different absolute configurations at C9-C12 ( 1 : 9 S , 10 R , 11 S , 12 S ; 2 : 9 R , 10 S , 11 R , 12 R ). Compounds 1 and 2 exhibited potent blocking activities against Kv1.5 with IC 50 values of 1.22 ± 0.22 μM and 2.85 ± 0.29 μM, respectively.

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel whose α subunit is Kcnma1, and a voltage-dependent calcium channel, Cacna1d. It has also been suggested based on physiological and pharmacological evidence that a voltage-gated K channel is present in the basal membranes of the receptor cells which modulates synaptic transmitter release. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory β subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 using purified mRNA of homogenized electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the Bellono et al. RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent K channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels. The second ortholog is similar to Kv1.5 but has fewer N-terminal amino acids than other species. The sequence for Kv1.5 in the skate is very strongly aligned with the recently reported sequence for potassium channels in the electroreceptors of the cat shark, S. retifer, which also modulate synaptic transmission. The latter channel was designated as Kv1.3 in the initial report, but we suggest that these channels are actually orthologs of each other, and that Kv1.5 is the prevailing designation. We also found a beta subunit sequence (Kcnab2) which may co-assemble with one or both of the voltage gated channels. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of Kcnma1, and found a computer-generated assembly that contained a complete sequence of its β subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 is absent. The evolutionary origin of the newly described K channels and their subunits was studied by alignments with mammalian sequences, including human, and also those in related fish: the whale shark (R. typus), the ghost shark (C.milii), and (S. retifer). There are also orthologous K channels of the lamprey, which has electroreceptors. Tree building and bootstrap programs were used to confirm phylogenetic inferences. Further research should focus on the subcellular locations of these channels, their gating behavior, and the effects of accessory subunits on gating., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hydrocortisone exerts adverse effects on various organs, including the heart. This study investigated the still unclear effects of hydrocortisone on electrophysiological and biochemical aspects of cardiac excitation-contraction coupling. In guinea pigs' hearts, hydrocortisone administration reduced the QT interval of ECG and the action potential duration (APD). In guinea pig ventricular myocytes, hydrocortisone reduced contraction and Ca 2+ transient amplitudes. These reductions and the effects on APD were prevented by pretreatment with the protein kinase C (PKC) inhibitor staurosporine. In an overexpression system of Xenopus oocytes, hydrocortisone increased hERG K + currents and reduced Kv1.5 K + currents; these effects were negated by pretreatment with staurosporine. Western blot analysis revealed dose- and time-dependent changes in PKCα/βII, PKCε, and PKCγ phosphorylation by hydrocortisone in guinea pig ventricular myocytes. Therefore, hydrocortisone can acutely affect cardiac excitation-contraction coupling, including ion channel activity, APD, ECG, Ca 2+ transients, and contraction, possibly via biochemical changes in PKC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Atrial fibrillation (AF) is a major cause of stroke, heart failure, sudden death and cardiovascular morbidity. The Kv1.5 potassium channel conducts the IKur current and has been demonstrated to be predominantly expressed in atrial versus ventricular tissue. Blockade of Kv1.5 has been proven to be an effective approach to restoring and maintaining sinus rhythm in preclinical models of AF. In the clinical setting, however, the therapeutic value of this approach remains an open question. Herein, we present synthesis and optimization of a novel series of 1,2-bis(aryl)ethane-1,2-diamines with selectivity for Kv1.5 over other potassium ion channels. The effective refractory period in the right atrium (RAERP) in a rabbit PD model was investigated for a selection of potent and selective compounds with balanced DMPK properties. The most advanced compound (10) showed nanomolar potency in blocking Kv1.5 in human atrial myocytes and based on the PD data, the estimated dose to man is 700 mg/day. As previously reported, 10 efficiently converted AF to sinus rhythm in a dog disease model., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

BMS-919373 is a highly functionalized quinazoline under investigation as a selective, potent I Kur current blocker. By utilizing the aminomethylpyridine side chain at C-4, a selective C-H functionalization at C-5 was invented, enabling the efficient synthesis of this molecule. The strategy of leveraging this inherent directing group allowed the synthesis of this complex heterocycle in only six steps from commodity chemicals. The scope of the C-H activation was further investigated, and the generality of the transformation across a series of bicyclic aromatic heterocycles was explored.

Background: Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents., Conclusions: Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

Three kinds of new water-soluble polysaccharides (FA, FB and FC) were isolated from wild mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam by the classical method "water extraction and alcohol precipitation" and purified by column chromatography. The Mw of FA, FB and FC ranged from 5690 Da to 38,340 Da. The three polysaccharide fractions in the fruiting body were mainly composed of 4 kinds of monosaccharides, including glucose, galactose, mannose, and arabinose, among which glucose and galactose were the major monosaccharides. The FTIR and NMR spectroscopy indicated that the skeleton of three fractions composed of a (1→4)-α-D-glycosidic backbone containing α-D-mannopyranose. In vitro anti-hypoxia activity data showed that three polysaccharide fractions possessed a significant effect on inhibiting PASM cells apoptosis under hypoxia. Among them, FC at the concentration of 200 µg/mL revealed a significant anti-hypoxia effect. These results revealed that the intracellular polysaccharides possessed potent anti-hypoxic activity, which might be related to inhibiting LDH and NADPH oxidase expression and promoting the formation of 5-hydroxytryptamine, dopamine, endothelins, acetylcholine. More importantly, FC showed good performance inducing KV1.5 expression and prohibiting KIR6.2 formation at protein level.

Key Points: The expression of miR-1 is increased in lungs from the Hyp/Su5416 PAH rat model. Pulmonary artery smooth muscle cells from this animal model are more depolarized and show decreased expression and activity of voltage-dependent potassium channel (Kv)1.5. miR-1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. Antagomir-1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition., Abstract: Impairment of the voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs are non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region region of specific mRNAs. The present study aimed to analyse the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of the patch clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells. A luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O 2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces pulmonary artery smooth muscle cell hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Background/aims: The phenylalkylamine class of L-type Ca 2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium., Methods: Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore., Results: Verapamil preferentially blocked hKv1.5 channel in its open state with IC 50 of 2.4±0.6 μM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516., Conclusion: Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel ( i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage., (© 2018 Lamothe et al.)

MK801 and ketamine, which are phencyclidine (PCP) derivative N-methyl-d-aspartate receptor (NMDAr) blockers, reportedly enhance the function of 5-hydroxytryptamine (HT)-2A receptors (5-HT 2A Rs). Both are believed to directly affect the pathogenesis of schizophrenia, as well as hypertension. 5-HT 2A R signaling involves the inhibition of Kv conductance. This study investigated the interaction of these drugs with Kv1.5, which plays important roles in 5-HT 2A R signaling and in regulating the excitability of the cardiovascular and nervous system, and the potential role of this interaction in the enhancement of the 5-HT 2A R-mediated response. Using isometric organ bath experiments with arterial rings and conventional whole-cell patch-clamp recording of Chinese hamster ovary (CHO) cells ectopically overexpressing Kv1.5, we examined the effect of ketamine and MK801 on 5-HT 2A R-mediated vasocontraction and Kv1.5 channels. Both ketamine and MK801 potentiated 5-HT 2A R-mediated vasocontraction. This potentiation of 5-HT 2A R function occurred in a membrane potential-dependent manner, indicating the involvement of ion channel(s). Both ketamine and MK801 rapidly and directly inhibited Kv1.5 channels from the extracellular side independently of NMDArs. The potencies of MK801 in facilitating the 5-HT 2A R-mediated response and blocking Kv1.5 were higher than those of ketamine. Our data demonstrated the direct inhibition of Kv1.5 channels by MK801/ketamine and indicated that this inhibition may potentiate the functions of 5-HT 2A Rs. We suggest that 5-HT 2A R-Kv1.5 may serve as a receptor-effector module in response to 5-HT and is a promising target in the pathogenesis of MK801-/ketamine-induced disease states such as hypertension and schizophrenia.

Background: Sudden unexplained nocturnal death syndrome (SUNDS) remains an autopsy negative entity with unclear etiology. Arrhythmia has been implicated in SUNDS. Mutations/deficiencies in intercalated disc components have been shown to cause arrhythmias. Human cardiomyopathy-associated 1 (XIRP1) and 3 (XIRP2) are intercalated disc-associated, Xin repeats-containing proteins. Mouse Xirp1 is necessary for the integrity of intercalated disc and for the surface expression of transient outward and delayed rectifier K + channels, whereas mouse Xirp2 is required for Xirp1 intercalated disc localization. Thus, XIRP1 and XIRP2 may be potentially causal genes for SUNDS., Methods and Results: We genetically screened XIRP genes in 134 sporadic SUNDS victims and 22 Brugada syndrome (BrS) cases in a Chinese Han population. We identified 16 rare variants (6 were in silico predicted as deleterious) in SUNDS victims, including a novel variant, XIRP2-E215K. There were also four rare variants (2 were in silico predicted as deleterious) detected in BrS cases, including a novel variant, XIRP2-L2718P. Interestingly, among these 20 variants, we detected 2 likely pathogenic variants: a nonsense variant (XIRP2-Q2875*) and a frameshift variant (XIRP2-T2238QfsX7). Analyzing available Xirp2 knockout mice, we further found that mouse hearts without Xirp2 exhibited prolonged PR and QT intervals, slow conduction velocity, atrioventricular conduction block, and an abnormal infranodal ventricular conduction system. Whole-cell patch-clamp detected altered ionic currents in Xirp2 -/- cardiomyocytes, consistent with the observed association between Xirp2 and Nav1.5/Kv1.5 in co-immunoprecipitation., Conclusions: This is the first report identifying likely pathogenic XIRP rare variants in arrhythmogenic disorders such as SUNDS and Brugada syndrome, and showing critical roles of Xirp2 in cardiac conduction., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Propofol blocks the voltage-gated human Kv1.5 (hKv1.5) channel by preferentially affecting in its open state. A previous mutational study suggested that several amino acids within the pore region of the hKv1.5 channel are involved in mediating the blocking action of propofol. The present investigation was undertaken to elucidate the predicted binding modes of propofol within the pore cavity of the open-state hKv1.5 channel, using computational docking and mutagenesis approaches. The docking simulation using a homology model of the hKv1.5 channel, constructed based on the crystal structure of the Kv1.2 channel, predicted that propofol was positioned at the base of the pore cavity of hKv1.5 channel, adjacent to 4 amino acids Thr479, Thr480, Val505, and Ile508, and formed arene-H interactions with Val505. The patch-clamp experiments on wild-type and mutant hKv1.5 channels constructed by site-directed mutagenesis revealed that the blocking potency of propofol was significantly reduced in T480A, V505A, and I508A but not in T479A mutants compared with wild-type hKv1.5 channel. These computational docking and experimental mutational analyses suggest that propofol is positioned at the base of the pore cavity and forms functional contact with Thr480, Val505, and Ile508 to directly block the hKv1.5 channel.

The voltage-gated K + channel has key roles in the vasculature and in atrial excitability and contributes to apoptosis in various tissues. In this study, we have explored its regulation by carbon monoxide (CO), a product of the cytoprotective heme oxygenase enzymes, and a recognized toxin. CO inhibited recombinant Kv1.5 expressed in HEK293 cells in a concentration-dependent manner that involved multiple signalling pathways. CO inhibition was partially reversed by superoxide dismutase mimetics and by suppression of mitochondrial reactive oxygen species. CO also elevated intracellular nitric oxide (NO) levels. Prevention of NO formation also partially reversed CO inhibition of Kv1.5, as did inhibition of soluble guanylyl cyclase. CO also elevated intracellular peroxynitrite levels, and a peroxynitrite scavenger markedly attenuated the ability of CO to inhibit Kv1.5. CO caused nitrosylation of Kv1.5, an effect that was also observed in C331A and C346A mutant forms of the channel, which had previously been suggested as nitrosylation sites within Kv1.5. Augmentation of Kv1.5 via exposure to hydrogen peroxide was fully reversed by CO. Native Kv1.5 recorded in HL-1 murine atrial cells was also inhibited by CO. Action potentials recorded in HL-1 cells were increased in amplitude and duration by CO, an effect mimicked and occluded by pharmacological inhibition of Kv1.5. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms. This regulation has important implications for diverse cellular functions, including excitability, contractility and apoptosis.

Redox-sensitive potassium channels consisting of the voltage-gated K + (K V ) channel pore subunit K V 1.5 regulate resting membrane potential and thereby contractility of vascular smooth muscle cells. Members of the K V 1 family associate with cytosolic auxiliary β subunits, which are members of the aldo-keto reductase (AKR) superfamily (AKR6A subfamily). The Kvβ subunits have been proposed to regulate Kv1 gating via pyridine nucleotide cofactor binding. However, the molecular identity of K V β subunits that associate with native K V 1.5 channels in the vasculature is unknown. Here, we examined mRNA and protein expression of K V β subunits and tested whether K V β isoforms interact with K V 1.5 channels in murine coronary arteries. We detected K V β1 (AKR6A3), K V β2 (AKR6A5) and K V β3 (AKR6A9) transcripts and K V β1 and K V β2 protein in left anterior descending coronary arteries by real time quantitative PCR and Western blot, respectively. In situ proximity ligation assays indicated abundant protein-protein interactions between K V 1.5/K V β1, K V 1.5/K V β2 and K V β1/β2 in coronary arterial myocytes. Confocal microscopy and membrane fractionation analyses suggest that arterial myocytes from K V β2-null mice have reduced abundance of sarcolemmal K V 1.5. Together, data suggest that in coronary arterial myocytes, K V 1.5 channels predominantly associate with K V β1 and K V β2 proteins and that K V β2 performs a chaperone function for K V 1.5 channels in arterial myocytes, thereby facilitating Kv1α trafficking and membrane localization., (Copyright © 2017 Elsevier B.V. All rights reserved.)