Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.     

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC₅₀ = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC₅₀s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC₅₀s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models.     

KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.     

A potent potassium channel blocker from Mesobuthus eupeus scorpion venom

Scorpion venom-derived peptidyl toxins are valuable pharmacological tools for investigating the structure–function relationship of ion channels. Here, we report the purification, sequencing and functional characterization of a new K⁺ channel blocker (MeuKTX) from the venom of the scorpion Mesobuthus eupeus. Effects of MeuKTX on ten cloned potassium channels in Xenopus oocytes were evaluated using two-electrode voltage-clamp recordings. MeuKTX is the orthologue of BmKTX (α-KTx3.6), a known Kv1.3 blocker from the scorpion Mesobuthus martensii, and classified as α-KTx3.13. MeuKTX potently blocks rKv1.1, rKv1.2 and hKv1.3 channels with 50% inhibitory concentration (IC₅₀) of 203.15 ± 4.06 pM, 8.92 ± 2.3 nM and 171 ± 8.56 pM, respectively, but does not affect rKv1.4, rKv1.5, hKv3.1, rKv4.3, and hERG channels even at 2 μM concentration. At this high concentration, MeuKTX is also active on rKv1.6 and Shaker IR. Our results also demonstrate that MeuKTX and BmKTX have the same channel spectrum and similar pharmacological potency. Analysis of the structure–function relationships of α-KTx3 subfamily toxins allows us to recognize several key sites which may be useful for designing toxins with improved activity on hKv1.3, an attractive target for T-cell mediated autoimmune diseases.     

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca²⁺-activated K⁺ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.     

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.     

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

Mutations in the KCNA1 gene associated with episodic ataxia type-1 syndrome impair heteromeric voltage-gated K(+) channel function.

Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.     

Identification of an intermolecular disulfide bond in barley hemoglobin

The present study was designed to investigate properties of ion channels in undifferentiated rabbit mesenchymal stem cells (MSCs) from bone marrow using whole-cell patch-clamp and RT-PCR techniques. It was found that three types of outward currents were present in rabbit MSCs, including an inward rectifier K(+) current (I(Kir)), a noise-like Ca(2+)-activated K(+) current (I(KCa)) co-present with delayed rectifier K(+) current (IK(DR)). I(Kir) was inhibited by Ba(2+), while I(KCa) was inhibited by paxilline (a blocker of big conductance I(KCa) channels) and clotrimazole (an inhibitor of intermediate conductance I(KCa) channels). IK(DR) exhibited a slow inactivation, "U-shaped" voltage-dependent inactivation, and slow recovery from inactivation, and the current was inhibited by tetraethylammonium or 4-aminopyridine. RT-PCR revealed the molecular identities for the functional ionic currents, including Kir1.1 (possibly responsible for I(Kir)), KCa1.1 and KCa3.1 (possibly responsible for I(KCa)), and Kv1.2, Kv2.1, and Kv2.2 (possibly responsible for IK(DR)). These results demonstrate for the first time that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and IK(DR)) are present in rabbit MSCs from bone marrow.     

Multiple Kv1.5 targeting to membrane surface microdomains

Surface expression of voltage-dependent K(+) channels (Kv) has a pivotal role in leukocyte physiology. Although little is known about the physiological role of lipid rafts, these microdomains concentrate signaling molecules and their ion channel substrates. Kv1.3 associates with Kv1.5 to form functional channels in macrophages. Different isoform stoichiometries lead to distinct heteromeric channels which may be further modulated by targeting the complex to different membrane surface microdomains. Kv1.3 targets to lipid rafts, whereas Kv1.5 localization is under debate. With this in mind, we wanted to study whether heterotetrameric Kv1.5-containing channels target to lipid rafts. While in transfected HEK-293 cells, homo- and heterotetrameric channels targeted to rafts, Kv1.5 did not target to rafts in macrophages. Therefore, Kv1.3/Kv1.5 hybrid channels are mostly concentrated in non-raft microdomains. However, LPS-induced activation, which increases the Kv1.3/Kv1.5 ratio and caveolin, targeted Kv1.5 back to lipid rafts. Moreover, Kv1.5 did not localize to low-buoyancy fractions in L6E9 skeletal myoblasts, which also coexpress both channels, heart membranes or cardiomyocyes. Coexpression of a Cav3(DGV)-mutant confined Kv1.5 to Cav3(DGV)-vesicles of HEK cells. Contrarily, coexpression of Kvbeta2.1 impaired the Kv1.5 targeting to raft microdomains in HEK cells. Our results indicate that Kv1.5 partnership interactions are underlying mechanisms governing channel targeting to lipid rafts.     

敬钊毒素-XI与突变体R3A-JZTX-XI的合成、复性及其药理活性鉴定

表达于b-胰岛细胞上的Kv2.1钾通道电流负责动作电位的复极化,从而调节胰岛素的分泌,是治疗2型糖尿病的有效作用靶点。敬钊毒素-XI(JZTX-XI) 是从敬钊缨毛蛛Chilobrachys jingzhao粗毒中分离纯化到的一种新型的肽类神经毒素,能够抑制非洲爪蟾卵母细胞上表达的Kv2.1钾通道电流。为了研究JZTX-XI的结构与功能关系,用芴甲氧羰基 (Fomc) 固相多肽合成方法合成了野生型JZTX-XI和突变体R3A-JZTX-XI,结合反相HPLC和质谱对不同条件下的氧化复性结果进行检测,从而得     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

KCNE beta subunits determine pH sensitivity of KCNQ1 potassium channels.

BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. Methods: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.     

Crosslinking of CD44 on human osteoblastic cells upregulates ICAM-1 and VCAM-1

Cancer-induced cachexia affects most advanced cancer patients. It is characterized by anorexia, profound metabolic dysfunctions, and severe neurological disorders. Here we show that voltage-gated potassium channel (Kv) expression is impaired in the brain of tumor-bearing animals. Expression of both delayed rectifier (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv4.2) and A-type potassium channels (Kv1.4, Kv3.3, Kv3.4) was greatly down-regulated in brain from animals bearing a Yoshida AH-130 ascites hepatoma. The possible compensatory mechanisms (Kv1.4/Kv4.2), expression of redundant genes (Kv3.1/Kv3.3) and heteromultimeric channel formation (Kv2.1/Kv9.3) were also affected. The high circulating levels of TNFalpha and the reduced expression of the anti-apoptotic protein Bcl-XL found in the brain of tumor-bearing animals indicate that this response could be mediated by an increase in brain cell death due to apoptosis. The results suggest that brain function is impaired during cancer cachexia, and may account for the cancer-induced anorectic response and other neurological alterations.     

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.     

alpha subunit compositions of Kv1.1-containing K+ channel subtypes fractionated from rat brain using dendrotoxins.

K+ channels from the Kv1 subfamily contain four alpha-subunits and the combinations (from Kv1.1-1.6) determine susceptibility to dendrotoxin (DTX) homologues. The subunit composition of certain subtypes in rat brain was investigated using DTXk which only interacts with Kv1.1-containing channels and alphaDTX (and its closely related homologue DTXi) that binds preferentially to Kv1. 2-possessing homo- or hetero-oligomers. Covalent attachment of [125I]DTXk bound to channels in synaptic membranes unveiled subunits of Mr = 78 000 and 96 000. Immunoprecipitation of these solubilized and dissociated cross-linked proteins with IgG specific for each of the alpha-subunits identified Kv1.1, 1.2 and 1.4; this led to assemblies of Kv1.1/1.2 and 1.1/1.4 being established. Kv1. 2-enriched channels, purified from rat brain by chromatography on immobilized DTXi, contained Kv1.1, 1.2 and 1.6 confirming one of the above-noted pairs and indicating an additional Kv1.1-containing oligomer (Kv1.1/1.2/1.6); the notable lack of Kv1.4 excludes a Kv1. 1/1.2/1.4 combination. On the other hand, channels with Kv1.1 as a constituent, isolated using DTXk, possessed Kv1.4 in addition to those found in the DTXi-purified oligomers; this provides convergent support for the occurrence of the three combinations established above but adds a possible fourth (Kv1.1/1.4/1.6), though this was not confirmed. Moreover, sequential purification on DTXi and DTXk resins yielded channels containing only Kv1.1/1.2 but with an apparent predominance of Kv1.1, reaffirming the latter multimer.     

The Ca2+-Activated K+ Channel KCa3.1 as a Potential New Target for the Prevention of Allograft Vasculopathy

Allograft vasculopathy (AV) remains one of the major challenges to the long-term functioning of solid organ transplants. Although its exact pathogenesis remains unclear, AV is characterized by both fibromuscular proliferation and infiltration of CD4⁺ memory T cells. We here tested whether two experimental immunosuppressants targeting K⁺ channels might be useful for preventing AV. PAP-1 inhibits the voltage-gated Kv1.3 channel, which is overexpressed on CCR7⁻ memory T cells and we therefore hypothesize that it should suppress the memory T cell component of AV. Based on its previous efficacy in restenosis and kidney fibrosis we expected that the KCa3.1 blocker TRAM-34 would primarily affect smooth muscle and fibroblast proliferation and thus reduce intimal hyperplasia. Using immunohistochemistry we demonstrated the presence of Kv1.3 on infiltrating T cells and of KCa3.1 on lymphocytes as well as on proliferating neointimal smooth muscle cells in human vasculopathy samples and in a rat aorta transplant model developing chronic AV. Treatment of PVG rats receiving orthotopically transplanted aortas from ACI rats with TRAM-34 dose-dependently reduced aortic luminal occlusion, intimal hyperplasia, mononuclear cell infiltration and collagen deposition 120 days after transplantation. The Kv1.3 blocker PAP-1 in contrast did not reduce intima hyperplasia despite drastically reducing plasma IFN-γ levels and inhibiting lymphocyte infiltration. Our findings suggest that KCa3.1 channels play an important role in the pathogenesis of chronic AV and constitute an attractive target for the prevention of arteriopathy.