细胞外La~(3+)对内向整流钾通道(IRK1)的阻断作用

本文采用双微电极电压钳 (TEV)法研究细胞外La3+对非洲爪蟾卵母细胞表达的内向整流钾通道(IRK1)的阻断作用。细胞外La3+浓度分别为 0 ,0 1,0 3,1,3和 10mmol/L ,K+浓度为 90mmol/L ,可见La3+对IRK1的瞬间电流 (施加电压后 1 5ms)具有La3+浓度依赖性、时间依赖性和电压依赖性阻断作用 ;阻断剂La3+对IRK1的门控特性和外向电流几乎无影响作用 ;细胞外加La3+后反转电位没有变化 ,因而IRK1对之不通透。细胞外La3+在减少IRK1电导的同时增加IRK1的归一化电导。三级指数拟合的结果表明 :拟合的时间常数不随La3+浓度的增减而增减 ,这表明细胞外La3+对IRK1的抑制作用可能通过了表面电荷机制或La3+在通道中的阻断位点在通道表面。因为La3+浓度较低时 ,阻断的效力与La3+浓度较高时的差异不大 ,所以La3+不会通过表面电荷机制进行IRK1阻断的 ,因此La3+是IRK1的一种快速开通道阻断剂 ,其阻断位点在通道表面     

细胞外Ba^2＋对双基因内向整流钾通道的阻断作用

目的：研究Ｂａ＾２＋对非洲爪蟾卵母细胞表达的双基因内向整流钾通道（ＩＲＫ１－Ｔ）的阻断作用。方法：采用双微电极电压钳（ＴＥＶ）法。结果：细胞外Ｂａ＾２＋浓度分别为１，３，１０和１００μｍｏｌ／Ｌ，Ｋ＾＋浓度为９０ｍｍｏｌ／Ｌ，可见Ｂａ＾２＋的阻断作用对ＩＲＫ１－Ｔ的瞬间电流（施加电压后１ｍｓ）具有浓度依赖性、时间信赖３性和电压信赖性；快速开通道阻断剂Ｂａ＾２＋对ＩＲＫ１－Ｔ的通道开关特性几乎无影响作用，ＩＲＫ１－Ｔ对之不能透。三级指数拟合表明：细胞外Ｂａ＾２＋低浓度（１和３　ｍｏｌ／Ｌ）时，Ｂａ＾２＋与Ｋ＾＋相互竞争同一结合位点，随着Ｂａ＾２＋浓度的增加，时间常数不增加但拟合的权数却浓度依赖性增加，所以失活过程随Ｂａ＾２＋浓度的增加越来越快；细胞外Ｂａ＾２＋高浓度（１０和１００μｍｏｌ／Ｌ）时，时间常数随Ｂａ＾     

乙酰胆碱对离体豚鼠心房肌动作电位及其脱敏的影响

目的 :研究迷走神经递质乙酰胆碱 （ACh）对离体豚鼠心房肌细胞动作电位 （AP）的影响及其对心房肌的脱敏 ,探讨脱敏可能发生的机制。方法 :采用标准玻璃微电极细胞内记录方法 ,观察不同浓度 （0 .0 1,0 .1,1μm ol/ L） ACh对心房肌细胞 AP的影响及对动作电位时程 （APD）和收缩力的脱敏现象 ,并观察了受体或通道水平的阻断剂阿托品、氯化铯 （Cs Cl）对 ACh所致的心房肌 APD脱敏的影响。结果 :10 .0 1,0 .1,1μm ol/ L ACh分别缩短 APD的变化率为 （6 .0 2± 1.0 4） % ,（14.2 0± 3.79） % ,（30 .2 0± 3.6 5 ） %。 2 0 .0 1μmol/ L ACh对心房肌细胞没有脱敏 ;0 .1μm ol/ L ACh对心房肌细胞 APD有轻微脱敏 ,脱敏持续时间为 1m in;而 1μmol/ L ACh对心房肌细胞 APD脱敏明显 ,脱敏持续时间为 5 m in。 31μmol/ L 阿托品与 2 0 m mol/ L Cs Cl并不能阻断 1μmol/ L ACh对心房肌细胞APD的脱敏。结论 :ACh缩短 APD的作用随浓度增大而增大 ;ACh对离体豚鼠心房肌细胞 APD的脱敏有浓度依赖性与时间依赖性 ;脱敏的发生可能与毒蕈碱型胆碱能受体及 Ik,ACh电流有关。     

急性淋巴细胞白血病用甲氨喋啶治疗的动力学研究

目的 :根据不同年龄 ,采用两种不同大剂量甲氨喋啶 2 4h持续静滴下的稳态血药浓度及毒性反应。方法 :分别于甲氨喋啶静滴开始后 1、6、2 3、44、6 8h点采集静脉血 2 ml及 1、2 4h点脑脊液 2 m l,应用免疫偏振荧光法检测血清与脑脊液 MTX浓度。结果 :9例老年组采用 2 g· M- 2 剂量 ,在 1、6、2 3、44、6 8h点血清 MTX浓度分别为 5 6 .97± 4.7μmol/ L、40 .46± 7.0 6μmol/ L、17.31± 9.76μm ol/ L、0 .91± 1.34μm ol/ L、0 .2 5± 0 .2 2μmol/L ;在 1、2 4h点脑脊液 MTX浓度分别为 0 .38± 0 .2 4μm ol/ L、2 .0 7± 0 .76μmol/ L。 16例中青年组采用 3 g· M- 2剂量 ,各小时点血清 MTX浓度分别为 82 .15± 12 .13μmol/ L、6 2 .33± 10 .0 6μm ol/ L、14.75± 7.0 6μm ol/ L、1.71± 1.6 8μm ol/ L、0 .45± 0 .44 μmol/ L;脑脊液 MTX浓度分别为 0 .5 4± 0 .35 μmol/ L、1.96± 0 .5 2 μm ol/ L。结论 :此两种剂量能达到急性淋巴细胞白血病 (AL L)细胞聚谷氨酸盐 MTX饱和所需的剂量 ,毒性反应是可以接受的。     

克罗卡林和JTV-506对大鼠离体心脏心功能的影响

目的 :对比两种不同的 ATP敏感性钾通道 （KATP通道 ）开放剂克罗卡林 （CR）和 JTV- 5 0 6 （JTV）对大鼠离体心脏心功能的影响及缺血后的心脏保护作用。方法 :用含不同药物浓度的灌注液灌注心脏并将心脏进行完全缺血 2 5min,再灌注 30 m in,观察各组心功能的变化。结果 :1～ 2 0μm ol/ L CR及 JTV均能显著增加冠脉灌注量 ,1μm ol/ L的 CR和 JTV较对照组均有改善缺血后心功能的作用 ,其作用可完全被格列本脲所阻断。结论 :两药均有明显冠脉扩张作用 ,大剂量应用可降低心脏收缩功能 ,用不影响心功能的剂量两者均有对缺血后心脏的保护作用 ,此作用与ATP敏感性钾通道开放有关。     

Kvβ1.3亚基显著影响DPO-1对表达在卵母细胞上的Kvl.5通道的阻断作用

目的:研究Kvβ1.3亚基和Kv1.5共表达时,对表达在非洲爪蟾卵母细胞的Kv1.5通道DPO-1的阻断作用的影响。方法:在非洲爪蟾卵母细胞上异源表达克隆Kv1.5及Kvβ1.3通道基因,用双电极电压钳技术记录全细胞电流,检测药物对Kv1.5通道及Kv1.5+Kvβ1.3共表达通道电流的影响。结果:DPO-1以电压、频率及浓度依赖方式抑制Kv1.5+Kvβ1.3共表达通道的电流。Kvβ1.3亚基存在时,DPO-1的阻断效应明显减弱,DPO-1阻断的IC50由(0.77士0.12)μmol/L显著增加至(47.21士5.18)μmol/L,增加了约60倍(P<0.01)。结论:Kvβ1.3亚基显著抑制DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用,但不改变其电压、频率及浓度依赖性,可能机制是Kvβ1.3亚基与DPO-1相互竞争Kv1.5孔区内部的某些结合位点。     

Kvβ1.3亚基显著影响DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用

目的:研究Kvβ1.3亚基和Kv1.5共表达时,对表达在非洲爪蟾卵母细胞的Kv1.5通道DPO-1的阻断作用的影响。方法:在非洲爪蟾卵母细胞上异源表达克隆Kv1.5及Kvβ1.3通道基因,用双电极电压钳技术记录全细胞电流,检测药物对Kv1.5通道及Kv1.5+Kvβ1.3共表达通道电流的影响。结果:DPO-1以电压、频率及浓度依赖方式抑制Kv1.5+Kvβ1.3共表达通道的电流。Kvβ1.3亚基存在时,DPO-1的阻断效应明显减弱,DPO-1阻断的IC50由(0.77士0.12)μmol/L显著增加至(47.21士5.18)μmol/L,增加了约60倍(P0.01)。结论:Kvβ1.3亚基显著抑制DPO-1对表达在卵母细胞上的Kv1.5通道的阻断作用,但不改变其电压、频率及浓度依赖性,可能机制是Kvβ1.3亚基与DPO-1相互竞争Kv1.5孔区内部的某些结合位点。     

替米沙坦对表达在卵母细胞上Kv1.5通道的抑制效应

目的:研究替米沙坦对表达在卵母细胞上的克隆人类Kv1.5通道的作用,探讨其在心脏复极中的潜在效应。方法:在非洲爪蟾卵母细胞上异源表达克隆人类Kv1.5通道基因,使用双电极电压钳技术记录全细胞电流,检测药物对Ikur电流的影响。结果:替米沙坦以电压依赖性和浓度依赖性方式抑制Kv1.5通道电流,且对峰电流及1.5s末端电流的抑制效应不同,在1μmol/L浓度下,抑制效应分别达到(7.75±2.39)和(52.64±3.77),其半抑制浓度(IC50)分别为(2.25±0.97)μmol/L和(0.82±0.39)μmol/L。替米沙坦对通道的稳态失活没有显著改变,在对照条件下,V1/2的值为(14.47±3.71)mV,斜坡因子k为(23.24±3.86)mV;在1μmol/L替米沙坦作用下,V1/2和k的值分别为(14.38±4.62)mV和(26.26±5.04)mV(n=6,P>0.05)。同时,替米沙坦显著加速了Kv1.5通道的失活。在对照条件下,Kv1.5通道的失活慢时间常数是(693.74±23.16)ms,在应用1μmol/L替米沙坦后,其失活的慢时间常数下降为(523.85±10.28)ms(n=5,P<0.05)。结论:替米沙坦在临床有效浓度范围内能显著抑制表达在卵母细胞上的Ikur电流,提示它兼有选择性阻滞Kv1.5通道的作用。     

厄贝沙坦对电压依赖性的Kv1.3和Kv1.5通道电流的阻断作用

目的通过观察厄贝沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨厄贝沙坦对此类通道的阻断可能具有的临床作用。方法使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度厄贝沙坦灌流对其电流的影响。结果①厄贝沙坦浓度依赖性的阻断Kv1.3通道,阻断的IC50是2.46μmol/L,且阻断具有电压依赖性。②厄贝沙坦浓度依赖性的阻断Kv1.5通道,阻断的IC50是0.47μmol/L,且阻断具有显著的电压依赖性。结论厄贝沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一;而对开放状态的Kv1.5的阻断可能是其具备减少心房颤动发生率的作用机制之一。     

生长素对人脐静脉内皮细胞增殖和迁移的影响及机制

目的观察生长素对血管紧张素Ⅱ所致的脐静脉内皮细胞增殖和迁移的影响。方法采取不同浓度的血管紧张素Ⅱ(10-9~10-6mol/L)刺激脐静脉内皮细胞24 h,获取最佳浓度(10-6mol/L)后用此浓度的血管紧张素Ⅱ分别刺激脐静脉内皮细胞0、6、12和24 h,观察脐静脉内皮细胞的增殖和迁移;并观察生长素(10-9~10-6mol/L)预处理2 h后与10-6mol/L血管紧张素Ⅱ共同孵育24 h和10-6mol/L生长素预处理0、0.5、1和2 h后与10-6mol/L血管紧张素Ⅱ共同孵育24 h对脐静脉内皮细胞增殖和迁移的影响。加入丝裂原活化蛋白激酶/细胞外信号调节激酶信号通路阻滞剂PD98059(25μmol/L)、生长激素促分泌素受体1 a阻断剂([Dys3]GHRP-6 25μmol/L)预处理人脐静脉内皮细胞,观察其对生长素影响血管紧张素Ⅱ诱导的内皮细胞增殖和迁移的作用。水溶性四氮唑8法测细胞增殖,Transwell小室测细胞迁移,免疫印迹法测细胞中细胞外信号调节激酶1/2、磷酸化细胞外信号调节激酶1/2蛋白表达。结果血管紧张素Ⅱ呈浓度和时间依赖性促进内皮细胞迁移和增殖。生长素抑制血管紧张素Ⅱ引起的内皮细胞迁移和增殖呈浓度和时间依赖性。PD98059(25μmol/L)预处理可以抑制血管紧张素Ⅱ促内皮细胞增殖作用,[Dys3]GHRP-6(25μmol/L)预处理阻断生长素对内皮细胞迁移和增殖的抑制作用。生长素可减少磷酸化细胞外信号调节激酶1/2的表达。结论生长素可抑制血管紧张素Ⅱ诱导的内皮细胞增殖和迁移,其机制涉及细胞外信号调节激酶1/2途径。     

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K(+) channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K(+) selective, whereas those with two or three are nonselective and permeate Na(+) and K(+) equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K(+) channels is essential for highly selective and efficient permeation of K(+) ions.     

Autonomic Regulation of Voltage-Gated Cardiac Ion Channels

Altering voltage-gated ion channel currents, by changing channel number or voltage-dependent kinetics, regulates the propagation of action potentials along the plasma membrane of individual cells and from one cell to its neighbors. Functional increases in the number of cardiac sodium channels (Na_V1.5) at the myocardial sarcolemma are accomplished by the regulation of caveolae by β adrenergically stimulated G-proteins. We demonstrate that Na_V1.5, Ca_V1.2a, and K_V1.5 channels specifically localize to isolated caveolar membranes, and to punctate regions of the sarcolemma labeled with caveolin-3. In addition, we show that Na_V1.5, Ca_V1.2a, and K_V1.5 channel antibodies label the same subpopulation of isolated caveolae. Plasma membrane sheet assays demonstrate that Na_V1.5, Ca_V1.2a, and K_V1.5 cluster with caveolin-3. This may have interesting implications for the way in which adrenergic pathways alter the cardiac action potential morphology and the velocity of the excitatory wave.     

Structure and regulation of L-type calcium channels a current assessment of the properties and roles of channel subunits

L-type Ca channels are complex heteromultimeric proteins that play important roles in the cardiovascular system. Recent studies have revealed new insights into how the pore-forming ₁ subunits interact with accessory subunits to produce functional Ca channels. The function of L-type Ca channels is often regulated by receptor-mediated signal transduction events that are thought to result in the phosphorylation of proteins that comprise the Ca channels. Although the molecular events underlying phosphorylation based regulation have been intensely investigated with the use of electrophysiological approaches, surprisingly few details are known about the biochemical events involved, and many questions remain unanswered. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:265–273).     

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP(3) and Ca(2+) have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca(2+) dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca(2+) alone from the FRET changes evoked by both ligands together. A mutant IP(3)R containing a single amino acid substitution at K508, which is critical for IP(3) binding, did not exhibit this bell-shaped Ca(2+) dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca(2+) sensor, resulted in ～10-fold reduction in the Ca(2+) dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP(3)R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca(2+) binding affinity, as the mechanism underlying the IP(3)-dependent regulation of the bell-shaped relationship between the IP(3)R activity and cytosolic Ca(2+).     

心肌缺血预适应中的离子通道

缺血预适应调动机体内源性抗损伤能力，保护缺血缺氧的组织细胞，是近年来心血管领域研究的热点之一。本该对心肌缺血预适应中的离子通道、三磷酸腺苷敏感性钾通道（KATP通道）、L-型钙道道（L-Ca^2 通道）、体积调节性氯通道（Cl vol通道）的特性、作用及其机制作一综述。     

ATP-sensitive potassium channels and myocardial ischemia: Why do they open?

Summary There is evidence that the ATP-sensitive potassium channel opens, at least during the early stages of myocardial ischemia, despite relatively high ATP levels. Thus, channel opening may partially contribute to potassium efflux and accumulation of extracellular potassium, but probably much more profoundly to electrical abnormalities associated with ischemia, including the development of lethal arrhythmias. Several factors are discussed that may promote a significant open-channel probability of the channel, in spite of relatively high levels of ATP. It is argued that, even with a very low open probability, the magnitude of total membrane current carried by these channels may be substantial (comparable to other potassium currents) because of the high density and conductance of the ATP-sensitive potassium channel. Finally, it is shown how the ATP-sensitive potassium channel may play a role in various tissue types, ranging from the physiological to the pathophysiological. This potassium channel is therefore increasingly targeted for drug development and research.     

Endothelial‐smooth muscle cell interactions in the regulation of vascular tone in skeletal muscle

The SMCs of skeletal muscle arterioles are intricately sensitive to changes in membrane potential. Upon increasing luminal pressure, the SMCs depolarize, thereby opening VDCCs, which leads to contraction. Mechanisms that oppose this myogenic tone can involve voltage‐dependent and independent dilator pathways, and can be endothelium‐dependent or independent. Of particular interest are the pathways leading to hyperpolarization of SMCs, as these can potentially evoke both local and conducted dilation. This review focuses on three agonists that cause local and conducted dilation in skeletal muscle: ACh, ATP, and KCl. The mechanisms for the release of these agonists during motor nerve stimulation and/or hypoxia, and their actions to open either Ca²⁺‐activated K⁺ channels (K_Ca) or inwardly rectifying K⁺ channels (K_IR) are described. By causing local and conducted dilation, each agonist has the ability to improve skeletal muscle blood flow during exercise and ischemia.     

T-Type Calcium Channel Blockade in the Management of Chronic Ischemic Heart Disease

The T-Type calcium channel offers a new therapeutic target for teatment of patients with cardiovascular disease. Mibefradil, a T channel blocker, produces heart rate slowing and coronary vasodilatation but without the negative inotropic effect commonly seen when L-Type channel blockers are used. The present study shows Mibefradil prevents ischemic episodes that are and are not preceded by an increase in heart rate. Although Mibefradil has been withdrawn because of multiple drug interactions, new T-Type calcium channel blockers are under development.