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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).)

The objective of the study was to investigate the expression levels of potassium voltage-gated channel subfamily A member 5 (KCNA5), connexin 43 (Cx43), and connexin 40 (Cx40) in the left atrial appendage of patients with atrial fibrillation (AF) and the interactions between them. We gathered tissue samples from patients with persistent AF and sinus rhythm and used fluorescence quantitative polymerase chain reaction to evaluate messenger RNA (mRNA) changes of KCNA5, Cx43, and Cx40. Then, we studied the protein levels of KCNA5, Cx43, and Cx40 by immunofluorescence and western blot analysis and the interactions between these proteins were identified by immunoprecipitation and immunofluorescence colocation, respectively. Compared with the control group, the mRNA and protein levels of KCNA5, Cx43, and Cx40 in the AF group were decreased and the positive expression of KCNA5, Cx43, and Cx40 protein was also decreased by immunofluorescence staining in the AF group. In addition, immunoprecipitation and immunofluorescence colocation revealed that KCNA5 was coexpressed with Cx43 and Cx40 proteins. The expressions of KCNA5, Cx43, and Cx40 were substantially downregulated in the myocardium of patients with AF and KCNA5 interacted with Cx43 and Cx40 proteins, respectively., (© 2022 International Federation for Cell Biology.)

Ethnopharmacology Relevance: In folkloric medicine of many cultures, one of the medical uses of Valeriana officinalis Linn is to treat heart-related disease. Recently, it was shown that the ethanol extracts from V. officinalis could effectively prevent auricular fibrillation, and 8-hydroxypinoresinol-4-O-β-D-glucoside (HPG) from the extracts is one of the two active compounds showing antiarrhythmia activities., Aim of the Study: The human Kv1.5 channel (hKv1.5) has potential antiarrhythmia activities, and this study arms at investigating the current blocking effects of HPG on hKv1.5 channel., Material and Methods: HPG was obtained from V. officinalis extracts, and hKv1.5 channels were expressed in HEK 293 cells. HPG was perfused while recording the current through hKv1.5 channels. Patch-clamp recording techniques were used to study the effects of HPG at various concentrations (10 μM, 30 μM, and 50 μM) on hKv1.5 channels., Results: The present study demonstrated that HPG inhibited hKv1.5 channel current in a concentration-dependent manner; the higher the concentration, the greater is the inhibition at each depolarization potential. During washout, the channels did not full recover indicating that the un-coupling between HPG and hKv1.5 channels is a slow process., Conclusion: HPG may be an effective and safe active ingredient for AF having translational potential., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The dysregulation of K + channels is a hallmark of pulmonary arterial hypertension (PAH). Herein, the channelome was analyzed in lungs of patients with PAH in a public transcriptomic database. Sixty six (46%) mRNA encoding cationic channels were dysregulated in PAH with most of them downregulated (83%). The principal component analysis indicated that dysregulated cationic channel expression is a signature of the disease. Changes were very similar in idiopathic, connective tissue disease and congenital heart disease associated PAH. This analysis 1) is in agreement with the widely recognized pathophysiological role of TASK1 and K V 1.5, 2) supports previous preliminary reports pointing to the dysregulation of several K + channels including the downregulation of K V 1.1, K V 1.4, K V 1.6, K V 7.1, K V 7.4, K V 9.3 and TWIK2 and the upregulation of K Ca 1.1 and 3) points to other cationic channels dysregulated such as Kv7.3, TALK2, Ca V 1 and TRPV4 which might play a pathophysiological role in PAH. The significance of other changes found in Na + and TRP channels remains to be investigated., (© 2021. The Author(s).)

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.)

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.

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.)

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda , whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G ( 1 ) and neo-debromoaplysiatoxin H ( 2 ) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G ( 1 ) (3.6 mg) and neo-debromoaplysiatoxin H ( 2 ) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC 50 ) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC 50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

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.)

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: 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.)

The increase in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K + (K V ) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca 2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca 2+ influx through receptor-operated and store-operated Ca 2+ channels (SOC). Upon Ca 2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca 2+ entry. Activity of K V channels, like KCNA5/K V 1.5 and KCNA2/K V 1.2, contributes to regulating membrane potential, and inhibition of K V channels results in membrane depolarization that increases [Ca 2+ ] cyt by opening voltage-dependent Ca 2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca 2+ influx through SOC channels. Extracellular Ca 2+ -mediated activation of CaSR increases [Ca 2+ ] cyt in CASR -transfected HEK293 cells. Treatment of CASR -transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca 2+ ] cyt . Moreover, CaSR-mediated increase in [Ca 2+ ] cyt was significantly augmented in cells co-transfected with CASR and STIM2 . CaSR activation results in STIM2 clustering in CASR/STIM2 -cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K + currents in KCNA5 - and KCNA2 -transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca 2+ ] cyt by enhancing store-operated Ca 2+ entry and inhibits KCNA5/K V 1.5 and KCNA2/K V 1.2, ultimately leading to voltage-activated Ca 2+ entry.

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.)

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.

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.

Objective: We have previously described that changes in the expression of Kv channels associate to phenotypic modulation (PM), so that Kv1.3/Kv1.5 ratio is a landmark of vascular smooth muscle cells phenotype. Moreover, we demonstrated that the Kv1.3 functional expression is relevant for PM in several types of vascular lesions. Here, we explore the efficacy of Kv1.3 inhibition for the prevention of remodeling in human vessels, and the mechanisms linking the switch in Kv1.3 /Kv1.5 ratio to PM. Approach and Results: Vascular remodeling was explored using organ culture and primary cultures of vascular smooth muscle cells obtained from human vessels. We studied the effects of Kv1.3 inhibition on serum-induced remodeling, as well as the impact of viral vector-mediated overexpression of Kv channels or myocardin knock-down. Kv1.3 blockade prevented remodeling by inhibiting proliferation, migration, and extracellular matrix secretion. PM activated Kv1.3 via downregulation of Kv1.5. Hence, both Kv1.3 blockers and Kv1.5 overexpression inhibited remodeling in a nonadditive fashion. Finally, myocardin knock-down induced vessel remodeling and Kv1.5 downregulation and myocardin overexpression increased Kv1.5, while Kv1.5 overexpression inhibited PM without changing myocardin expression., Conclusions: We demonstrate that Kv1.5 channel gene is a myocardin-regulated, vascular smooth muscle cells contractile marker. Kv1.5 downregulation upon PM leaves Kv1.3 as the dominant Kv1 channel expressed in dedifferentiated cells. We demonstrated that the inhibition of Kv1.3 channel function with selective blockers or by preventing Kv1.5 downregulation can represent an effective, novel strategy for the prevention of intimal hyperplasia and restenosis of the human vessels used for coronary angioplasty procedures.

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.)

Background: Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide and in need of novel potential diagnostic biomarkers for early discovery., Methods: We conducted a two-step study. We first employed bioinformatics on data from The Cancer Genome Atlas to obtain potential biomarkers and then experimentally validated some of them on our clinical samples. Our aim was to find a methylation alteration common to all clusters, with the potential of becoming a diagnostic biomarker in CRC., Results: Unsupervised clustering of methylation data resulted in four clusters, none of which had a known common genetic or epigenetic event, such as mutations or methylation. The intersect among clusters and regulatory regions resulted in 590 aberrantly methylated probes, belonging to 198 differentially expressed genes. After performing pathway and functional analysis on differentially expressed genes, we selected six genes: CEP55, FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5, for further experimental validation on our own clinical samples. In silico analysis demonstrated that CEP55 was hypomethylated in 98.7% and up-regulated in 95.0% of samples. Genes FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 97.9, 81.1, 80.3, 98.4 and 94.0%, and down-regulated in 98.3, 98.9, 98.1, 98.1 and 98.6% of samples, respectively. Our experimental data show CEP55 was hypomethylated in 97.3% of samples and down-regulated in all samples, while FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 100.0, 90.2, 100.0, 99.1 and 100.0%, and down-regulated in 68.0, 76.0, 96.0, 95.2 and 84.0% of samples, respectively. Results of in silico and our experimental analyses showed that more than 97% of samples had at least four methylation markers altered., Conclusions: Using bioinformatics followed by experimental validation, we identified a set of six genes that were differentially expressed in CRC compared to normal mucosa and whose expression seems to be methylation dependent. Moreover, all of these six genes were common in all methylation clusters and mutation statuses of CRC and as such are believed to be an early event in human CRC carcinogenesis and to represent potential CRC biomarkers.

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 Intrauterine growth retardation ( IUGR ) is related to pulmonary artery hypertension in adults, and mi croRNA -206 (miR-206) is proposed to affect the proliferation and apoptosis of pulmonary artery smooth muscle cells ( PASMC s) via post-transcriptional regulation. Methods and Results In an IUGR rat model, we found that the expression and function of potassium voltage-gated channel subfamily A member 5 (Kv1.5) in PASMC s was inhibited, and pulmonary artery hypertension was exaggerated after chronic hypoxia ( CH ) treatment as adults. micro RNA expression was investigated in PASMC s from 12-week-old male IUGR rats with CH by microarray, polymerase chain reaction, and in situ hybridization. The expression levels of Kv1.5 in primary cultured PASMC s and pulmonary artery smooth muscle from IUGR or control rats were evaluated with and without application of an miR-206 inhibitor. Right ventricular systolic pressure, cell proliferation, luciferase reporter assay, and I K v were also calculated. We found increased expression of miR-206 in resistance pulmonary arteries of IUGR rats at 12 weeks compared with newborns. Application of an miR-206 inhibitor in vivo or in vitro increased expression of Kv1.5 α-protein and KCNA 5. Also, decreased right ventricular systolic pressure and cell proliferation were observed in PASMC s from 12-week-old control and IUGR rats after CH , while inhibitor did not significantly affect control and IUGR rats. Conclusions These results suggest that expression of Kv1.5 and 4-aminopyridine (Kv channel special inhibitor)-sensitive Kv current were correlated with the inhibition of miR-206 in PA rings of IUGR - CH rats and cultured IUGR PASMC s exposed to hypoxia. Thus, miR-206 may be a trigger for induction of exaggerated CH-pulmonary artery hypertension of IUGR via Kv1.5.

Background: The influence of 5-HTT, BMPR2, EDN1, ENG, KCNA5 genes polymorphisms on susceptibility of pulmonary arterial hypertension remains uncertain. This meta-analysis is conducted for further study., Methods: We conducted a literature search on PubMed and ISI web of science databases for searching relevant articles until November 2017. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. A total of 17 articles with 2631 PAH subjects and 5139 controls were included in the final meta-analysis. Statistical software Stata13.0 was used for data-analysis., Results: A significant relationship was found between the 5-HTT L/S polymorphism and PAH in all the genetic models [LL vs. SS: OR = 1.60, 95% CI, 1.11-2.32; LS vs. SS: OR = 1.55, 95% CI, 1.10-2.21; (LS + LL) vs. SS: OR = 1.56, 95% CI, 1.13-2.17; L vs. S: OR = 1.32, 95% CI, 1.08-1.62]. There were also associations of the SERT L/S polymorphism with IPAH and PAH in COPD [IPAH L/S: OR = 1.26, 95% CI, 1.01-1.57; PAH in COPD L/S: OR = 1.42, 95% CI, 1.04-1.94]. In addition, the results showed a statistically significant association between EDN1 rs5370 polymorphism and the risk of PAH in all the genetic models [TT vs. GG: OR = 3.32, 95% CI, 1.30-8.51; TG vs. GG: OR = 2.68, 95% CI, 1.54-4.66; (TG + TT) vs. GG: OR = 2.82, 95% CI, 1.69-4.71; T vs. G: OR = 2.43, 95% CI, 1.60-3.68]. However, the significant association was not found between BMPR2 rs1061157, KCNA5 rs10744676, ENG rs3739817 polymorphisms and the risk of PAH (all p > 0.05)., Conclusions: 5-HTT L/S polymorphism and END1 rs5370 polymorphism were correlated with significantly increased risk of PAH. Moreover, L allele in 5-HTT gene increased susceptibility to IPAH and PAH in COPD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.)

Molecular dynamics simulations are employed to determine the inhibitory mechanisms of three drugs, 5-(4-phenoxybutoxy)psoralen (PAP-1), vernakalant, and flecainide, on the voltage-gated K + channel Kv1.5, a target for the treatment of cardiac arrhythmia. At neutral pH, PAP-1 is neutral, whereas the other two molecules carry one positive charge. We show that PAP-1 forms stable dimers in water, primarily through hydrophobic interactions between aromatic rings. All three molecules bind to the cavity between the Ile508 and Val512 residues from the four subunits of the channel. Once bound, the drug molecules are flexible, with the average root-mean-square fluctuation being between 2 and 3 Å, which is larger than the radius of gyration of a bulky amino acid. The presence of a monomeric PAP-1 causes the permeating K + ion to dehydrate, thereby creating a significant energy barrier. In contrast, vernakalant blocks the ion permeation primarily via an electrostatic mechanism and, therefore, must be in the protonated and charged form to be effective.

Pulmonary artery hypertension (PAH) is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis)-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia), caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH., (Copyright © 2017. Published by Elsevier B.V.)

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.

A recent study has identified six novel genetic variations (D322H, E48G, A305T, D469E, Y155C, P488S) in KCNA5 (encoding Kv1.5 which carries the atrial-specific ultra-rapid delayed rectifier current, I Kur ) in patients with early onset of lone atrial fibrillation. These mutations are distinctive, resulting in either gain-of-function (D322H, E48G, A305T) or loss-of-function (D469E, Y155C, P488S) of I Kur channels. Though affecting potassium channels, they may modulate the cellular active force and therefore atrial mechanical functions, which remains to be elucidated. The present study aimed to assess the inotropic effects of the identified six KCNA5 mutations on the human atria. Multiscale electromechanical models of the human atria were used to investigate the impact of the six KCNA5 mutations on atrial contractile functions. It was shown that the gain-of-function mutations reduced active contractile force primarily through decreasing the calcium transient (CaT) via a reduction in the L-type calcium current (I CaL ) as a secondary effect of modulated action potential, whereas the loss-of-function mutations mediated positive inotropic effects by increased CaT via enhancing the reverse mode of the Na + /Ca 2+ exchanger. The 3D atrial electromechanical coupled model predicted different functional impacts of the KCN5A mutation variants on atrial mechanical contraction by either reducing or increasing atrial output, which is associated with the gain-of-function mutations or loss-of-function mutations in KCNA5, respectively. This study adds insights to the functional impact of KCNA5 mutations in modulating atrial contractile functions., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Levo-tetrahydropalmatine (l-THP) exerts various pharmacological effects on neural and cardiac tissues and K + channel can be one of its multiple targets. The rapidly activating Kv1.5 channel is expressed in a variety of tissues including atrial cells and hippocampal neurons, and has an essential role in tuning the action potential and excitability in those cells. The aim of current study is to explore whether there are the possible effects of l-THP on Kv1.5 channels expressed in HEK293 cells. Superfusion of l-THP led to a dose-dependent blockage of Kv1.5 currents with an IC 50 value of 53.2μM. This blocking effect was substantially attenuated in mutant H452G rather than R476V and R476Y, suggesting a specific binding site in the outer mouth region. In addition, the properties of Kv1.5 channel kinetics were markedly altered by l-THP. Treatment with l-THP resulted in a potential left shift of the inactivation curve, with the half-maximum inactivation potential (V 1/2 ) of 4.5mV in control and -12.8mV in 50μM l-THP. Our data reveal that l-THP can exert an inhibitory effect on the delayed rectifier Kv1.5 channels expressed in HEK293 cells. These lines of evidence provided an insight to understand the possible effects exerted by l-THP on relative tissues., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability., Methods and Results: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect., Conclusion: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology)

A recent experimental study investigating patients with lone atrial fibrillation identified six novel mutations in the KCNA5 gene. The mutants exhibited both gain- and loss-of-function of the atrial specific ultra-rapid delayed rectifier K+ current, IKur. The aim of this study is to elucidate and quantify the functional impact of these KCNA5 mutations on atrial electrical activity. A multi-scale model of the human atria was updated to incorporate detailed experimental data on IKur from both wild-type and mutants. The effects of the mutations on human atrial action potential and rate dependence were investigated at the cellular level. In tissue, we assessed the effects of the mutations on the vulnerability to unidirectional conduction patterns and dynamics of re-entrant excitation waves. Gain-of-function mutations shortened the action potential duration in single cells, and stabilised and accelerated re-entrant excitation in tissue. Loss-of-function mutations had heterogeneous effects on action potential duration and promoted early-after-depolarisations following beta-adrenergic stimulation. In the tissue model, loss-of-function mutations facilitated breakdown of excitation waves at more physiological excitation rates than the wild-type, and the generation of early-after-depolarisations promoted unidirectional patterns of excitation. Gain- and loss-of-function IKur mutations produced multiple mechanisms of atrial arrhythmogenesis, with significant differences between the two groups of mutations. This study provides new insights into understanding the mechanisms by which mutant IKur contributes to atrial arrhythmias. In addition, as IKur is an atrial-specific channel and a number of IKur-selective blockers have been developed as anti-AF agents, this study also helps to understand some contradictory results on both pro- and anti-arrhythmic effects of blocking IKur.

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

Aim of the Study  Nuclear factor of activated T-cell (NFATc2), a Ca 2+ /calcineurin-dependent transcription factor, is reported to be activated in human and animal pulmonary hypertension (PH). KV1.5, a voltage-gated K + (KV) channel, is expressed in pulmonary artery smooth muscle cells (PASMC) and downregulated in PASMC in patients and animals with PH. Furthermore, activation of NFATc2 downregulates expression of KV1.5 channels, leading to excessive PASMC proliferation. The aim of this study was to investigate the pulmonary vascular expression of NFATc2 and KV1.5 in rats with nitrofen-induced congenital diaphragmatic hernia (CDH). Materials and Methods  After ethical approval, time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day 9 (D9). When sacrificed on D21, the fetuses ( n  = 22) were divided into CDH and control groups. Using quantitative real-time polymerase chain reaction and western blotting, we determined the gene and protein expression of NFATc2 and KV1.5. Confocal microscopy was used to detect both proteins in the pulmonary vasculature. Results  Relative mRNA levels of NFATc2 were significantly upregulated and KV1.5 levels were significantly downregulated in CDH lungs compared with controls ( p  < 0.05). Western blotting confirmed the imbalanced pulmonary protein expression of both proteins. An increased pulmonary vascular expression of NFATc2 and a diminished expression of KV1.5 in CDH lungs compared with controls were seen in confocal microscopy. Conclusions  This study demonstrates for the first time an altered gene and protein expression of NFATc2 and KV1.5 in the pulmonary vasculature of nitrofen-induced CDH. Upregulation of NFATc2 with concomitant downregulation of KV1.5 channels may contribute to abnormal vascular remodeling resulting in PH in this model., (Georg Thieme Verlag KG Stuttgart · New York.)

Aims: Palmitate, a common saturated free fatty acid, induces endothelial apoptosis in vitro in culture endothelial cells and in vivo in type 2 diabetes mellitus (T2DM) patients. The present study aimed to investigate whether Kv1.5 regulates palmitate-induced endothelial apoptosis and endothelial dysfunction in T2DM., Main Methods: In vitro experiments were carried out in primary human HUVECs. Apoptosis was analyzed by flow cytometry. Cell viability was determined by Cell Counting Assay Kit-8. The siRNA transfection was employed to knockdown Kv1.5 protein expression. Intracellular and mitochondrial ROS, and mitochondrial membrane potential were detected using fluorescent probes. Male C57BL/6 mice fed with high-sucrose/fat diet were injected with streptozotocin (35mg/kg body weight) to establish T2DM animal model., Key Findings: We found that palmitate-induced endothelial apoptosis was parallel to a significant increase in endogenous Kv1.5 protein expression in endothelial cells. Silencing of Kv1.5 with siRNA reduced palmitate-induced endothelial apoptosis, intracellular ROS generation, mitochondrial ROS generation and membrane potential (Δψ m ) alteration and cleaved caspase-3 protein expression; while increased cell viability and ratio of Bcl-2/Bax. Furthermore, we observed that Kv1.5 protein expression increased in endothelial cells of thoracic aorta of T2DM mice. Silencing of Kv1.5 significantly improved the endothelium-dependent vasodilation in thoracic aortic rings of T2DM mice., Significance: These results demonstrate that suppression of Kv1.5 protects endothelial cells against palmitate-induced apoptosis via inhibiting mitochondria-mediated excessive ROS generation and apoptotic signaling pathway, suggesting that Kv1.5 may serve as a therapeutic target of treatment for endothelial dysfunction induced by palmitate and lipid metabolism in T2DM patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human Kv1.5 channels (hKv1.5) conduct the ultra-rapid delayed rectifier potassium current (I Kur ), which plays an important role in action potential repolarization of atrial myocytes. The present study was undertaken to examine the effects of acidic pH on hKv1.5 wild-type (WT) and its pore mutant channels heterologously expressed in Chinese hamster ovary (CHO) cells using site-directed mutagenesis combined with whole-cell patch-clamp technique. Both extracellular and intracellular acidifications equally and reversely reduced the amplitude of hKv1.5 currents. The extracellular acidification significantly shifted the voltage dependence of current activation to more depolarized potentials and accelerated deactivation kinetics of the current. The ancillary β subunits Kvβ1.3 and Kvβ1.2, known to modify the pharmacological sensitivities of hKv1.5, enhanced the extracellular proton-induced inhibitory effect on hKv1.5 current. In addition, several mutants (T462C, T479A, T480A, and I508A) exhibited significantly higher sensitivity to acidic pH-induced inhibition compared with WT channel, whereas the inhibitory effect of acidic pH was markedly reduced in H463G mutant. These observations indicate that (1) extracellular acidification modifies hKv1.5 gating and activity, (2) β subunits and several residues (T462, T479, T480, and I508) play critical roles in determining the sensitivity of the channel to acidic exposure, and (3) H463 may be a critical sensor for the channel inhibition by extracellular protons.

Objective: To identify the disease-causing gene of a family with upper limb predominant, slowly progressive amyotrophic lateral sclerosis (ALS), which was diagnosed as flail arm syndrome (FAS)., Methods: After causation of 24 known ALS genes was excluded by targeted next-generation sequencing, whole-exome sequencing was applied in the FAS family. Cellular localization of mutant hnRNPA1 was examined in transfected HeLa cells. An additional 251 Chinese patients with ALS (including 7 sporadic FAS) underwent mutation screening of hnRNPA1., Results: We detected a novel missense mutation in hnRNPA1, c.862/1018C>T (p.P288S/P340S), which cosegregated with disease in the FAS family. The residue is highly conserved across species and exists in the encoded PY nuclear localization signal of hnRNPA1 protein. Mutant hnRNPA1 showed altered intracellular localization, resulting in formation of cytoplasmic inclusions that colocalized with stress granules in transfected cells. Further mutation screening of hnRNPA1 in additional patients with FAS and typical ALS detected 2 rare variants with unknown significance. These variants lie in the prion-like domain of hnRNPA1 long isoform, which was detected exclusively in the CNS., Conclusions: Our results suggest that hnRNPA1 is the causative gene in the family with flail arm ALS. This further expanded the disease phenotype of hnRNPA1 mutations., (© 2016 American Academy of Neurology.)

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Voltage-gated potassium (Kv) channels are involved in the proliferation and transformation of mammary epithelial cells. They are thought to be related to the development of breast carcinoma, although the exact role they play in this event remains unclear. In this study, we investigated whether the expression and function of Kv channels is associated with Caveolin-1 (Cav-1, a principal component of caveolae) in different cell lines. We found that expression of Cav-1 correlated with the expression of Kv channels in mammary epithelial cells (MCF10A, MCF10A-ST1, and MCF7), and silencing of Cav-1 inhibited the expression of KCNA5 (voltage-gated shaker-related subfamily A, member 5). Immunofluorescence analysis indicated the colocalization of KCNA5 with Cav-1, whereas immunoprecipitation suggested a possible interaction between the two proteins. Overall, our finding indicated that KCNA5 protein may interact with Cav-1, thereby contributing to the proliferation and early transformation of mammary cells.

Hyperaldosteronism is associated with an increased prevalence of atrial fibrillation (AF). Mutations in KCNE3 have been associated with AF, and Kcne3(-/-) mice exhibit hyperaldosteronism. In this study, we used recently developed Kcne3(-/-) mice to study atrial electrophysiology with respect to development of aldosterone-dependent AF. In invasive electrophysiology studies, Kcne3(-/-) mice displayed a reduced atrial effective refractory period (AERP) and inducible episodes of paroxysmal AF. The cellular arrhythmogenic correlate for AF predisposition was a significant increase in atrial Kv currents generated by the micromolar 4-aminopyridine-sensitive Kv current encoded by Kv1.5. Electrophysiological alterations in Kcne3(-/-) mice were aldosterone dependent and were associated with increased Rab4, -5, and -9-dependent recycling of Kv1.5 channels to the Z-disc/T-tubulus region and lateral membrane via activation of the Akt/AS160 pathway. Treatment with spironolactone inhibited Akt/AS160 phosphorylation, reduced Rab-dependent Kv1.5 recycling, normalized AERP and atrial Kv currents to the wild-type level, and reduced arrhythmia induction in Kcne3(-/-) mice. Kcne3 deletion in mice predisposes to AF by a heretofore unrecognized mechanism-namely, increased aldosterone-dependent Kv1.5 recycling via Rab GTPases. The findings uncover detailed molecular mechanisms underpinning a channelopathy-linked form of AF and emphasize the inevitability of considering extracardiac mechanisms in genetic arrhythmia syndromes.-Lisewski, U., Koehncke, C., Wilck, N., Buschmeyer, B., Pieske, B., Roepke, T. K. Increased aldosterone-dependent Kv1.5 recycling predisposes to pacing-induced atrial fibrillation in Kcne3(-/-) mice., (© FASEB.)

Kv1.5 channels mediate the ultra-rapidly activating delayed rectifier potassium current (IKur), which is important for atrial repolarization. It has been shown that cell-surface Kv1.5 channels are sensitive to cleavage by the extracellular serine protease, proteinase K (PK). Here, we investigated the effects of extracellular proteolytic digestion on the function of Kv1.5 channels stably expressed in HEK 293 cells. Our data demonstrate that PK treatment cleaved mature membrane-bound (75kDa) Kv1.5 channels at a single locus in the S1-S2 linker, producing 42-kDa N-terminal fragments and 33-kDa C-terminal fragments. Interestingly, such PK treatment did not affect the Kv1.5 current (IKv1.5) recorded using the whole-cell patch clamp technique. Analysis of cell-surface proteins isolated using biotinylation indicated that the PK-generated N- and C-terminal fragments were both present in the plasma membrane. Co-immunoprecipitation (co-IP) experiments indicated that the N- and C-terminal fragments are no longer associated after cleavage. Furthermore, following PK digestion, the N- and C-fragments degraded at different rates. PK is frequently used as a tool to analyze cell-surface localization of membrane proteins, and cleavage of cell-surface channels has been shown to abolish channel function (e.g. hERG). Our data, for the first time, demonstrate that cleavage of cell-surface channels assessed by Western blot analysis does not necessarily correlate with an elimination of the channel activities., (Copyright © 2016 Elsevier B.V. All rights reserved.)