心房颤动与Kv1.5钾通道阻滞剂及其研究进展

心房颤动是临床常见的心律失常,药物是心房颤动的主要治疗方法。胺碘酮和心律平等药物虽然可以治疗和转复心房颤动,但长期应用会引起恶性心律失常和心脏外的副作用。抑制Kv1.5钾通道电流,可选择性延长心房肌动作电位时程及有效不应期,改善心房肌的电重构和组织重构。近年来关于Kv1.5钾通道及其阻滞剂的研究迅速发展并引起广泛关注。为进一步探讨Kv1.5钾通道是否可能成为心房颤动的治疗靶点,我们对目前相关研究进展作一综述。     

Kv1.5通道蛋白参与人脑胶质瘤细胞的自噬

脑胶质瘤是原发性颅内恶性肿瘤。患者的5年存活率不足1%。目前,除手术切除外,尚无有效的治疗手段。近年来发现,脑胶质瘤发病可能与多种钾离子通道的异常表达有关。自噬是膜包裹部分胞质和细胞内需降解的蛋白质、细胞器,并与溶酶体一起降解其所包裹内容物的生理过程。诱导胶质瘤细胞的自噬,促进其凋亡是肿瘤治疗的一种新策略。本室前期研究发现,电压依赖型钾通道1.5(Kv1.5)参与胞膜小窖标志蛋白质(caveolae,Cav-1)介导的多种肿瘤细胞的增殖和凋亡,但是否参与胶质瘤细胞的自噬并不清楚。本文首先利用不同浓度的K+通道阻断剂四乙胺(tetra-ethylammonium,TEA)、Kv通道阻断剂四氨基吡啶(4-amino-pyridine,4-AP)和Kv1.5通道特异性阻断剂DPO-1(diphenyl phosphine oxide-1)分别在不同时间,作用于人脑胶质瘤细胞U251,观察其对细胞存活的影响。发现DPO-1对U251细胞具有双向作用:低浓度促进存活,高浓度抑制存活。其中,1 mmol/L DPO-1处理6 h,可促进自噬相关蛋白质LC3的表达,而抑制mTOR信号蛋白质的磷酸化水平,表明Kv1.5通道可能参与胶质瘤细胞的自噬。然后,利用基因转染技术分别敲低和过表达Kv1.5通道的蛋白质水平,发现敲低Kv1.5通道蛋白,促进胶质瘤细胞的自噬,激活ERK信号通路,而过表达Kv1.5通道蛋白,则抑制胶质瘤细胞的自噬。进一步利用流式细胞技术观察细胞凋亡,发现改变Kv1.5通道蛋白的表达水平,可诱发细胞早期凋亡。提示Kv1.5通道参与人脑胶质瘤细胞的自噬过程。这为临床利用特异性Kv通道阻断剂靶向治疗胶质瘤提供了新的理论和实验依据。     

乙醇对大鼠心肌动作电位及人Kv1.5通道的影响

为了研究乙醇对心肌动作电位的作用及其机制,本实验采用标准玻璃微电极细胞内记录技术记录离体大鼠心肌细胞的动作电位(action potential,AP),采用全细胞膜片钳技术记录HEK293细胞上表达的人Kv1.5(human Kv1.5,hKv1.5)通道电流,观察6.25、12.5、25.0、50.0、100.0及...     

Myelinating Schwann cells determine the internodal localization of Kv1.1, Kv1.2, Kvβ2, and Caspr

We examined the localization of Caspr and the K⁺ channels Kv1.1 and Kv1.2, all of which are intrinsic membrane proteins of myelinated axons in the PNS. Caspr is localized to the paranode; Kv1.1, Kv1.2 and their β2 subunit are localized to the juxtaparanode. Throughout the internodal region, a strand of Caspr staining is flanked by a double strand of Kv1.1/Kv1.2/Kvβ2 staining. This tripartite strand apposes the inner mesaxon of the myelin sheath, and forms a circumferential ring that apposes the innermost aspect of Schmidt-Lanterman incisures. The localization of Caspr and Kv1.2 are not disrupted in mice with null mutations of the myelin associated glycoprotein, connexin32, or Kv1.1 genes. At all of these locations, Caspr and Kv1.1/Kv1.2/Kvβ2 define distinct but interrelated domains of the axonal membrane that appear to be organized by the myelin sheath.     

Cdk-mediated phosphorylation of the Kvβ2 auxiliary subunit regulates Kv1 channel axonal targeting

Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvβ2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvβ2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvβ2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvβ2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvβ2 to EB1.     

Structural determinants of the regulation of the voltage-gated potassium channel Kv2.1 by the modulatory α-subunit Kv9.3

Voltage-gated potassium (Kv) channels containing alpha-subunits of the Kv2 subfamily mediate delayed rectifier currents in excitable cells. Channels formed by Kv2.1 alpha-subunits inactivate from open- and closed states with both forms of inactivation serving different physiological functions. Here we show that open- and closed-state inactivation of Kv2.1 can be distinguished by the sensitivity to intracellular tetraethylammonium and extracellular potassium and lead to the same inactivated conformation. The functional properties of Kv2.1 are regulated by its association with modulatory alpha-subunits (Kv5, Kv6, Kv8, and Kv9). For instance, Kv9.3 changes the state preference of Kv2.1 inactivation by accelerating closed-state inactivation and inhibiting open-state inactivation. An N-terminal regulatory domain (NRD) has been suggested to determine the function of the modulatory alpha-subunit Kv8.1. However, when we tested the NRD of Kv9.3, we found that the functional properties of chimeric Kv2.1 channels containing the NRD of Kv9.3 (Kv2.1(NRD)) did not resemble those of Kv2.1/Kv9.3 heteromers, thus questioning the role of the NRD in Kv9 subunits. A further region of interest is a PXP motif in the sixth transmembrane segment. This motif is conserved among all alpha-subunits of the Kv1, Kv2, Kv3, and Kv4 subfamilies, whereas the second proline is not conserved in any modulatory alpha-subunit. Exchanging this proline in Kv2.1 for the corresponding residue of Kv9.3 resulted in channels (Kv2.1-P410T) that show all hallmarks of the regulation of Kv2.1 by Kv9.3. The effect prevailed in heteromeric channels following co-expression of Kv2.1-P410T with Kv2.1. These data suggest that the alteration of the PXP motif is an important determinant of the regulatory function of modulatory alpha-subunits.     

Substrate profiling and aldehyde dismutase activity of the Kvβ2 subunit of the mammalian Kv1 potassium channel

Voltage-dependent potassium channels (Kv) are involved in various cellular signalling processes by governing the membrane potential of excitable cells. The cytosolic face of these α subunit-containing channels is associated with β subunits that can modulate channel responses. Surprisingly, the β subunit of the mammalian Kv1 channels, Kvβ2, has a high level of sequence homology with the aldo–keto reductase (AKR) superfamily of proteins. Recent studies have shown that Kvβ2 can catalyze the reduction of aldehydes and, most significantly, that channel function is modulated when Kvβ2-bound NADPH is concomitantly oxidized. As a result, the redox chemistry of this subunit is crucial to understanding its role in K⁺ channel modulation. The present study has extended knowledge of the substrate profile of this subunit using a single turnover fluorimetric assay. Kvβ2 was found to catalyse the reduction of aromatic aldehyde substrates such as 2, 3 and 4-nitrobenzaldehydes, 4-hydroxybenzaldehyde, pyridine 2-aldehyde and benzaldehyde. The presence of an electron withdrawing group at the position para to the aldehyde in aromatic compounds facilitated reduction. Aliphatic aldehydes proved to be poor substrates. We devised a simple HPLC-based assay to identify Kvβ2 reaction products. Using this assay we showed, for the first time, that Kvβ2 can catalyze a slow aldehyde dismutation reaction using 4-nitrobenzaldehyde as substrate and have identified the products of this reaction. The ability of Kvβ2 to carry out both an aldehyde reduction and a dismutation reaction is discussed in the light of current thinking on the role of redox chemistry in channel modulation.     

ERK1/2对低氧大鼠肺动脉平滑肌细胞Kv1.5通道表达的影响

目的:探讨ERK1/2对低氧大鼠肺动脉平滑肌细胞(PASMCs)电压门控性钾离子通道(Kv1.5)表达的影响及其机制。方法:原代培养SD大鼠PASMCs,选3~6代PASMCs随机分组:1正常组(N);2低氧组(H);3DMSO组(HD);4U0126组(HU):10μmol/L U0126;5茴香霉素组(HA):10μmol/L茴香霉素。每组3皿细胞,正常组于常氧培养箱(5%CO2,37℃),其余各组均加入0.05%二甲基亚砜(DMSO)于低氧培养箱(5%CO2,2%O2,37℃),均培养60 h。采用RT-PCR和Western blot法测定PASMCs Kv1.5 mRNA和蛋白表达。结果:与N组相比,H、HD、HA组Kv1.5 mRNA和蛋白表达均明显降低(P0.05);较之H和HD组,HU组Kv1.5 mRNA和蛋白表达明显上升((P0.05),与HU组比较,HA组Kv1.5 mRNA和蛋白表达均明显降低(P0.05)。结论:低氧降低Kv1.5 mRNA和蛋白表达,U0126能够对抗低氧引起的Kv1.5通道的低表达,茴香霉素对低氧条件下Kv1.5通道表达无明显影响,但其表达仍显著低于常氧组,提示低氧可能通过干预ERK1/2信号通路抑制Kv1.5通道表达引起低氧性肺动脉高压。     

Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K⁺ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.     

Heteromerization of Arabidopsis Kv channel α-subunits: Data and prospects

Potassium translocation in plants is accomplished by a large variety of transport systems. Most of the available molecular information on these proteins concerns voltage-gated potassium channels (Kv channels). The Arabidopsis genome comprises nine genes encoding α-subunits of Kv channels. Based on knowledge of their animal homologues, and on biochemical investigations, it is broadly admitted that four such polypeptides must assemble to yield a functional Kv channel. The intrinsic functional properties of Kv channel α-subunits have been described by expressing them in suitable heterologous contexts where homo-tetrameric channels could be characterized. However, due to the high similarity of both the polypeptidic sequence and the structural scheme of Kv channel α-subunits, formation of heteromeric Kv channels by at least two types of α-subunits is conceivable. Several examples of such heteromeric plant Kv channels have been studied in heterologous expression systems and evidence that heteromerization actually occurs in planta has now been published. It is therefore challenging to uncover the physiological role of this heteromerization. Fine tuning of Kv channels by heteromerisation could be relevant not only to potassium transport but also to electrical signaling within the plant.Key words: heteromerization, voltage-gated channels, membrane potential     

Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic β-cells

Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic β-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic β-cells, suppression of glucose metabolism with low glucose concentrations of 2.8 mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to −10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0 mM but not 10 mM MgATP modulated the channel activity in a manner similar to that in β-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in β-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.     

慢性吸烟大鼠气道平滑肌大电导的钙激活钾通道和Kv1.5表达的变化

复制大鼠的慢性吸烟模型,采用气道反应性的测定、HE染色、免疫组织化学染色、原位杂交和免疫印迹实验等方法,观察吸烟对大鼠支气管平滑肌大电导的钙激活的钾通道(BKca)和电压依赖性延迟整流钾通道Kv1．5蛋白和mRNA表达的影响,以阐明吸烟引起的气道高反应性发病机制中钾通道表达变化的作用。结果显示：(1)慢性吸烟可降低大鼠大气道和小气道BKca和Kv1．5蛋白和mRNA表达;(2)大气道BKca的降低程度大于Kv1．5,小气道BKca和Kv1．5的降低程度无明显差异：(3)吸烟对全肺组织BKca和Kv1．5的蛋白表达无明显影响。上述结果提示,慢性吸烟可下调大鼠气道平滑肌钾通道BKca和Kv1．5的表达水平,是导致气道高反应的机制之一。     

Modulation of Closed?State Inactivation in Kv2.1/Kv6.4 Heterotetramers as Mechanism for 4?AP Induced Potentiation

The voltage−gated K⁺ (Kv) channel subunits Kv2.1 and Kv2.2 are expressed in almost every tissue. The diversity of Kv2 current is increased by interacting with the electrically silent Kv (KvS) subunits Kv5−Kv6 and Kv8−Kv9, into functional heterotetrameric Kv2/KvS channels. These Kv2/KvS channels possess unique biophysical properties and display a more tissue-specific expression pattern, making them more desirable pharmacological and therapeutic targets. However, little is known about the pharmacological properties of these heterotetrameric complexes. We demonstrate that Kv5.1, Kv8.1 and Kv9.3 currents were inhibited differently by the channel blocker 4−aminopyridine (4−AP) compared to Kv2.1 homotetramers. In contrast, Kv6.4 currents were potentiated by 4−AP while displaying moderately increased affinities for the channel pore blockers quinidine and flecainide. We found that the 4−AP induced potentiation of Kv6.4 currents was caused by modulation of the Kv6.4−mediated closed−state inactivation: suppression by 4−AP of the Kv2.1/Kv6.4 closed−state inactivation recovered a population of Kv2.1/Kv6.4 channels that was inactivated at resting conditions, i.e. at a holding potential of −80 mV. This modulation also resulted in a slower initiation and faster recovery from closed−state inactivation. Using chimeric substitutions between Kv6.4 and Kv9.3 subunits, we demonstrated that the lower half of the S6 domain (S6c) plays a crucial role in the 4−AP induced potentiation. These results demonstrate that KvS subunits modify the pharmacological response of Kv2 subunits when assembled in heterotetramers and illustrate the potential of KvS subunits to provide unique pharmacological properties to the heterotetramers, as is the case for 4−AP on Kv2.1/Kv6.4 channels.     

兔血管平滑肌延迟整流钾通道研究及其与克隆Kv1.5通道的电生理特性比较

本文用膜片箝全细胞技术比较了研究了单个兔肺动脉血管平滑肌细胞上延迟整流钾通道与克隆Ｋｖ１．５通道的电生理及药理学特性。将平滑肌细胞箝制在－４０ｍＶ,以１０ｍＶ的步跨阶跃去极化（０￣６０ｍＶ）可产生一系列快速上升的外向电流,几无衰减,其激活曲线的Ｖ１／２为２７．２ｍＶ。灌流液中加入１００ｍｍｏｌ／Ｌ和ＴＥＡ １ｍｍｏｌ／Ｌ ４ＡＰ,电流幅度均明显减小,细胞外Ｃａ＾２＋水平由１．５ｍｍｏｌ／Ｌ降至０．     

Structure–activity relationship exploration of Kv1.3 blockers based on diphenoxylate

Diphenoxylate, a well-known opioid agonist and anti-diarrhoeal agent, was recently found to block Kv1.3 potassium channels, which have been proposed as potential therapeutic targets for a range of autoimmune diseases. The molecular basis for this Kv1.3 blockade was assessed by the selective removal of functional groups from the structure of diphenoxylate as well as a number of other structural variations. Removal of the nitrile functional group and replacement of the C-4 piperidinyl substituents resulted in several compounds with submicromolar IC₅₀ values.     

Protein Kinase C-Mediated Phosphorylation of Kvβ2 in Adult Rat Brain

The phosphorylation of Kvβ2 was investigated by different protein kinases. Protein kinase A catalytic subunit (PKA-CS) yielded the greatest phosphorylation of recombinant Kvβ2 (rKvβ2), with limited phosphorylation by protein kinase C catalytic subunit (PKC-CS) and no detectable phosphorylation by casein kinase II (CKII). Protein kinase(s) from adult rat brain lysate phosphorylated both rKvβ2 and endogenous Kvβ. The PKA inhibitor, PKI 6-22, fully inhibited PKA-mediated phophorylation of rKvβ2 yet showed minimal inhibition of kinase activity present in rat brain. The inhibitor Gö 6983, that blocks PKCα, PKCβ, PKCγ, PKCδ and PKCζ activities, inhibited rKvβ2 phosphorylation by rat brain kinases, with no inhibition by Gö 6976 which blocks PKCα and PKCβΙ activities. Dose-response analysis of Gö 6983 inhibitory activity indicates that at least two PKC isozymes account for the kinase activity present in rat brain. Τhus, while PKA was the most active protein kinase to phosphorylate rKvβ2 in vitro, Kvβ2 phosphorylation in the rat brain is mainly mediated by PKC isozymes.     

Kv12.1 channels are not sensitive to GqPCR-triggered activation of phospholipase Cβ

K_v12.1 K⁺ channels are expressed in several brain areas, but no physiological function could be attributed to these subunits so far. As genetically-modified animal models are not available, identification of native K_v12.1 currents must rely on characterization of distinct channel properties. Recently, it was shown in Xenopus laevis oocytes that K_v12.1 channels were modulated by membrane PI(4,5)P₂. However, it is not known whether these channels are also sensitive to physiologically-relevant PI(4,5)P₂ dynamics. We thus studied whether K_v12.1 channels were modulated by activation of phospholipase C β (PLCβ) and found that they were insensitive to receptor-triggered depletion of PI(4,5)P₂. Thus, K_v12.1 channels add to the growing list of K⁺ channels that are insensitive to PLCβ signaling, although modulated by PI(4,5)P₂ in Xenopus laevis oocytes.     

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure,Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.