Novel Loss-of-Function KCNA5 Variants in Pulmonary Arterial Hypertension.

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.

Kv1.5 channels are regulated by PKC-mediated endocytic degradation.

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 (IKv1.5) 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.

Interaction of KCNA5, CX43, and CX40 proteins in the atrial muscle of patients with atrial fibrillation.

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.

Mechanical stretch increases Kv1.5 current through an interaction between the S1-S2 linker and N-terminus of the channel.

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 (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 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 IKv1.5.

Non-dioxin-like polychlorinated biphenyl 19 has distinct effects on human Kv1.3 and Kv1.5 channels.

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 IC50 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.

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

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 KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 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 KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 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 KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 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 KV1.5 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 KV1.5 channels in the plasma membrane.

Notch enhances Ca2+ entry by activating calcium-sensing receptors and inhibiting voltage-gated K+ channels.

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) 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 Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ 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 Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]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 [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.

MiR-3940-5p promotes granulosa cell proliferation through targeting KCNA5 in polycystic ovarian syndrome.

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.

Myocardin-Dependent Kv1.5 Channel Expression Prevents Phenotypic Modulation of Human Vessels in Organ Culture.

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.

Role of peroxisome proliferators-activated receptor-gamma in advanced glycation end product-mediated functional loss of voltage-gated potassium channel in rat coronary arteries.

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.

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes.

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 (IC50 = 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.

miR-1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries.

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% O2 ) 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.

The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.

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.

Identification of Verapamil Binding Sites Within Human Kv1.5 Channel Using Mutagenesis and Docking Simulation.

BACKGROUND/AIMS: The phenylalkylamine class of L-type Ca2+ 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 IC50 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.

The Potassium Channel Kv1.5 Expression Alters During Experimental Autoimmune Encephalomyelitis.

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.

Polysaccharides from the Edible Mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam Show Anti-hypoxia Activities in Pulmonary Artery Smooth Muscle Cells.

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.

Inhibition of Voltage-Gated K+ Channel Kv1.5 by Antiarrhythmic Drugs.

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.

In silico assessment of genetic variation in KCNA5 reveals multiple mechanisms of human atrial arrhythmogenesis.

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.

Interactions of Propofol With Human Voltage-gated Kv1.5 Channel Determined by Docking Simulation and Mutagenesis Analyses.

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.

In-silico investigations of the functional impact of KCNA5 mutations on atrial mechanical dynamics.

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, IKur) 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 IKur 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 (ICaL) 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+/Ca2+ 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.