去甲肾上腺素对缺氧心肌细胞钙通道的影响

目的 :研究去甲肾上腺素 ( NE)对缺氧心肌细胞钙通道的影响。方法 :运用全细胞膜片钳记录技术检测 NE对缺氧豚鼠心肌细胞钙通道电流 ( ICa)的影响。结果 :NE( 1× 10 - 8m ol/ L)可明显增加缺氧及缺氧 /再灌注心肌细胞 ICa( P <0 .0 1,<0 .0 5 ) ,使缺氧心肌细胞 ICa电流 -电压曲线下移 ;在缺氧状态及缺氧 /再灌注状态下 ,NE对心肌细胞 ICa的作用差异无显著性意义 ( P >0 .0 5 )。结论 :NE增加心肌细胞 ICa为其致缺血及缺血 /再灌注性心律失常机制之一     

颅痛定对单个豚鼠心室肌细胞缺氧后复氧早期L-Ca电流的影响

目的 :研究颅痛定 (左旋四氢巴马汀 ,L - tetrahy- dropalm atine,L - THP)对缺氧后复氧早期心室肌细胞 L - Ca电流的影响。方法 :使用全细胞膜片钳技术研究心室肌细胞 L - Ca电流的变化。结果 :经 2 0 m in的缺氧以及 5 min的复氧后 ,1,10 ,10 0 (μmol· L- 1 ) L - THP分别将 Ca- max电流从 (6 75 .75± 10 7.0 7) p A减少至 (6 45 .46±10 8.75 ) p A、(4 92 .0 8± 72 .84) p A、(376 .6 0± 71.35 ) p A ,P值分别为 >0 .0 5、<0 .0 5、<0 .0 1。结论 :L - THP能有效地减少缺氧后复氧早期心室肌细胞 L - Ca电流 ,这种作用无疑是其抗再灌注早期心律失常的电生理基础。     

心肌梗死后心肌T型钙通道基因表达及在心肌重塑中的作用

目的 :探讨心肌梗死后心肌重塑过程中T型钙通道基因表达的变化及其在心肌重塑中的作用。方法 :Wistar大鼠 18只 ,随机分为心肌梗死后 3h、2 1d组及假手术组 3组 ,每组各 6只 ,建立心肌梗死后心肌重塑模型。假手术组不结扎冠状动脉。采用RT PCR方法检测T型钙通道基因表达的变化 ;采用免疫组织化学方法染色和计算机图像分析方法检测Ⅰ型、Ⅲ型胶原和纤维连接蛋白 (FN)表达的变化 ;采用原子吸收光谱法测定心肌总Ca2 + 浓度。结果 :心肌梗死后 2 1d心肌重塑过程T型钙通道基因在邻近梗死区非梗死心肌表达 ,而远离梗死区心肌不表达 ;假手术组不出现表达。心肌Ⅰ型、Ⅲ型胶原 ,FN在梗死组心肌显著表达 ,与假手术组比较差异有显著性意义 (P <0 .0 5或 <0 .0 1)。心肌总Ca2 + 测定显示 ,梗死组比假手术组明显增加 (P <0 .0 5 )。结论 :T型钙通道与心肌梗死后心肌重塑密切相关。钙通道基因表达的意义可能在于增加心肌细胞膜钙通道密度。Ca2 + 经过钙通道进入细胞触发心肌重塑过程。提示 ,临床上开发和利用T型钙通道阻滞剂对防治心肌梗死后心肌重塑、心力衰竭和心律失常具有重要价值     

短期快速心房激动对L型钙通道mRNA表达的影响

目的 :观察短期快速心房激动对L型钙通道α1c亚基mRNA表达的影响。方法 :新西兰大耳白兔 36只 ,随机分为 6组 ,经颈内静脉切开置入电极导管 ,于右心房分别给予 0、0 .5、1、2、4、8h快速心房起搏 ,停止起搏后立即取右心房组织 ,应用半定量逆转录 聚合酶链式反应测定L型钙通道α1c亚基mRNA表达的相对水平 ,组间比较行t检验。结果 :0 .5～ 8h不同时点的快速心房起搏使L型钙通道α1c亚基mRNA表达水平 (分别为 0 .77±0 .0 3、0 .76± 0 .0 2、0 .77± 0 .0 5、0 .73± 0 .0 4、0 .6 7± 0 .0 5、0 .6 7± 0 .0 3)逐渐下调 ,致起搏后 4h较起搏前差异有显著性意义 (P <0 .0 5 )。结论 :短期快速心房激动使L型钙通道α1c亚基mRNA表达下调     

乙酰胆碱诱发的豚鼠心室肌反跳作用研究

观察乙酰胆碱 (ACh)诱发的豚鼠心室肌肌力及钙电流的反跳作用 ,并探讨其作用机制。采用豚鼠离体左室乳头肌观察 1μmol/LACh单独对心室肌收缩力 (Fc)的影响及在 10nmol/L异丙肾上腺素 (Iso)存在时对Fc的影响 ,并应用膜片钳全细胞记录方法分别观察 1μmol/LACh和 1μmol/LACh +10nmol/LIso及迅速洗脱ACh对豚鼠心室肌细胞L型钙通道电流 (ICa L)的影响。结果 :1μmol/LACh对心室肌Fc有直接抑制作用 ,抑制率为 30 .8%± 10 .7%(P <0 .0 5 ) ,而快速洗脱ACh后 ,Fc反跳性增强了 2 5 .5 %± 10 .3% (n =7,P <0 .0 1)。而在应用 10nmol/LIso后 1μmol/LACh对Fc有间接的抑制作用 ,快速冲洗ACh后亦引起Fc的反跳性增强 ,与Iso组相比增强了 2 7.1%±13.2 % (n =7,P <0 .0 1)。 1μmol/LACh对心室肌细胞基础峰电流无明显影响 (n =8,P >0 .0 5 ) ;而以基础ICa L峰值(931± 16 1pA)作对照 ,在加入 10nmol/LIso后 ,ICa L峰电流增强到 1889± 331pA(n =7,P <0 .0 1) ;再给予 10nmol/LIso +1μmol/LACh快速灌流 2min ,峰电流降低为 15 12± 2 0 2pA(P <0 .0 1) ,用含 10nmol/LIso的细胞外液快速洗脱ACh ,峰电流增强到 2 10 7± 2 0 5pA ,较Iso组反跳性增强了 15 .8%± 4 .0 % (n =7,P <0 .0 1)。结论 :ACh对豚鼠心室肌收缩     

熄风复遂汤联合钙通道抑制剂治疗急性脑梗死近期疗效研究

目的 :观察熄风复遂汤联合钙通道抑制剂治疗急性脑梗死(ACI)的疗效并探讨其作用机制。方法 :选择 75例符合诊断标准的病例 ,7d内起病的ACI病人 ,随机分为治疗组、对照 1组和对照 2组 ,每组 2 5例。治疗组以尼莫地平 10mg( 5 0ml)静脉输注 ,每日 1次 ,同时每日服 1剂熄风复遂汤 ,1个疗程后停用 5d ,继续第 2个疗程。对照 1组 :单用尼莫地平 ,剂量同治疗组。对照 2组 :单服中药。 3组均以 15d为一疗程。结果 :第 1个疗程、第 2个疗程后 ,治疗组与自身治疗前比较 ,NFD有统计学意义 (P <0 .0 5 ,P <0 .0 0 1) ;第 2疗程后 ,治疗组与对照 1组、对照 2组比较 ,NFD均有统计学意义 ;治疗组与对照 1组、对照 2组比较 ,有效率、显效率均有统计学意义 (P <0 .0 5 ,P <0 .0 0 1)。结论 :中药方剂熄风复遂汤联合钙通道抑制剂尼莫地平治疗ACI ,取得比单一用药更好的近期治疗效果 ,且副作用少。     

兔急性心肌梗死后梗死周边带心肌细胞L-型钙通道的变化

探讨L型钙通道在急性心肌梗死 (AMI)后室性心律失常发生中的作用及其机制。方法 :以开胸冠状动脉结扎法制备兔AMI模型 ,1周后处死动物分离心室肌细胞 ,采用全细胞膜片钳记录技术观察梗死周边缺血带心外膜心室肌细胞L型钙通道电流 (ICa L)的变化 ,以正常心肌ICa L为对照。结果 :AMI 1周时兔梗死周边区心室肌细胞L型钙电流受到抑制 ,其电流峰值由正常状态下的 - 5 .58± 1 .53pA/pF(对照组 ,n =1 0 )降至 - 3 .52± 0 .93pA/pF(AMI组 ,n=6) ,最大峰电流下降 2 9.1 % ,P <0 .0 5 ,I V曲线上移 ;其失活曲线左移 ,半数最大失活电位由 - 1 3 .1± 4 .2mV左移至 - 2 5 .9± 7.0mV ,P <0 .0 5 ,失活速度加快。结论 :AMI后 1周梗死周边带心外膜心室肌细胞L型钙通道受抑制 ,可能为AMI后室性心律失常发生的机制之一。     

兔急性心肌梗死后心室肌细胞L-钙离子通道电流的变化

目的 :研究急性心肌梗死 （AMI）后心室肌细胞钙离子通道电流的变化。方法 :采用结扎兔冠状动脉左前降支的方法建立 AMI动物模型 ,应用膜片钳全细胞记录方法 ,观察 AMI后 1周及 2月心外膜梗死区心肌细胞 L -钙通道电流 （ICa- L）的变化。结果 :1梗死后 1周组、2月组与对照组相比 ,I- V曲线上移。ICa- L电流密度峰值 （0 m V时 ）的比较显示 :对照组为 5 .6± 1.5 p A/ pf（n=10 ） ;梗死后 1周组为 3.5± 0 .9p A / pf（n=6 ） ,较对照组显著减小 ,P<0 .0 5 ;梗死后 2月组为 4 .8± 1.5 p A/ pf（n=11） ,较对照组减小 ,较梗死后 1周组增大 ,但均无统计学差异 ,P>0 .0 5。 2梗死后 1周组、梗死后 2月组与对照组相比 ,失活曲线明显左移 （即向超极化方向移动 ） ,以梗死后 1周组左移更加明显。梗死后 1周组半数失活电压 （V0 .5）为 - 2 6± 7m V（n=6 ） ,与对照组 （- 13± 4 m V ,n=8）比较相差显著 ,P<0 .0 5。梗死后 2月组半数失活电压 V0 .5为 - 2 1± 6 m V（n=8） ,与对照组比较无统计学差异 ,P>0 .0 5。结论 :AMI后1周梗死区心室肌细胞 ICa- L下降、钙通道动力学发生变化 ,在 AMI后 2月这种电生理异常有恢复趋势     

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+).     

Molecular structure of an open human KATP channel

K_ATP channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of K_ATP includes an inward-rectifier potassium channel (Kir) and an ABC transporter–like sulfonylurea receptor (SUR). Although structures of K_ATP have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic K_ATP (hK_ATP) structures with an open pore at 3.1- to 4.0-Å resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hK_ATP exhibits the unique (among inward-rectifier channels) property of PIP₂-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP₂.

K _ATP, a K⁺ channel that is gated by intracellular ATP and ADP (1–7), functions in many different cells including pancreatic β-cells (6), heart (8), skeletal muscle (9), smooth muscle (10), and neurons (11). By regulating K⁺ permeability as a function of cytoplasmic ATP and ADP concentrations, K_ATP links membrane electrical excitability to a cell’s energy budget (1). In pancreatic β-cells, this K_ATP-mediated link couples insulin secretion to serum glucose concentration (5, 6, 12–16). K_ATP is thus a pharmacological target for the treatment of type II diabetes (17–19).K_ATP consists of an inward-rectifier potassium channel (Kir) surrounded by four sulphonylurea receptors (SUR) that belong to the ABC transporter family (20–23) (Fig. 1A). Kir, a tetramer with four identical subunits, contains an ATP-binding site on the cytoplasmic domain (CTD) of each subunit (24–26). This site binds ATP with higher affinity than ADP (27). When ATP binds, pore closure is favored and thus the ATP site on Kir is referred to as inhibitory (1, 24). Each SUR subunit contains two adenosine nucleotide binding sites nestled in between two nucleotide binding domains (NBDs) (28, 29). These sites are formed when the NBDs engage each other (a process called dimerization) (28, 30, 31). One site, termed the degenerate site because it is incapable of mediating ATP hydrolysis, binds both ATP and ADP. The other, termed the consensus site, mediates ATP hydrolysis and favors ADP binding. Notably, and in contrast to most ABC transporters, Mg^2+-ADP alone is sufficient to dimerize the NBDs (32)—and when dimerization occurs, pore opening is favored (27). The opposing influence of ATP and ADP is central to the regulation of K_ATP gating in cells (1).Open in a separate windowFig. 1.Functional validation of purified human K_ATP (hKir6.2-hSUR1). (A) Locations of inhibitory (red) and activating (green) ATP and ADP in K_ATP. Kir subunit is colored in blue, and SUR subunit is colored in yellow. In all recordings, the membranes do not contain PIP₂. A total of 2 mM MgCl₂ was included in recording buffers. Currents are plotted according to physiological conventions such that inward current is negative. (B) Representative single-channel recording of reconstituted WT hK_ATP at two membrane voltages. Current levels for closed and one and two simultaneously opened channels are labeled as C, O1, and O2. (C) WT hK_ATP was activated by C8-PIP₂ and inhibited by ATP. Although not shown in the figure, ATP inhibition also occurs in the absence of PIP₂. (D) Locations of C166 (purple spheres) and G334 (cyan spheres) in the structural model of WT hKir6.2. Inhibitory ATP is colored in red. (E) hK_ATP (G334D_Kir) was activated by both ATP and ADP. (F) Representative single-channel recording of hK_ATP (C166S_Kir). Current levels for closed and one opened channel are labeled as C and O1. (G) Representative single-channel recording of hK_ATP (C166S_Kir, G334D_Kir). Current levels for closed and one to three simultaneously opened channels are labeled as C, O1, O2, and O3.Even though many molecular structures of K_ATP have been determined (32–39), we still cannot explain how ATP and ADP regulate the gate. All of the structures show the same closed conformation (32–39), so we cannot correlate conformational changes near the binding sites with those near the gate. In this paper, we describe a method for expressing and isolating a human pancreatic K_ATP (hK_ATP) complex composed of independent polypeptides. We show that hK_ATP channels in a reconstituted system exhibit physiological and pharmacological properties similar to those in cells. Then, through mutagenic alteration of the inhibitory ATP-binding site and a gate residue, we produce hK_ATP that exhibits high open probability and no ATP inhibition. Using single-particle cryo-electron miscroscopy (cryo-EM), we characterize hK_ATP with an open pore. From this structure, we correlate protein conformational changes that connect the ATP and ADP regulatory sites to the gate.     

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.     

普罗帕酮对豚鼠心室肌细胞离子通道活性的影响

应用膜片钳全细胞技术研究普罗帕酮对单个心室肌细胞膜离子通道的影响。结果表明：普罗帕酮灌流浓度为１×１０－６，６×１０－６Ｍ时，钠峰值电流分别下降３５．０％、５２．８％，Ｐ均＜０．０１；灌流浓度为１×１０－６，３×１０－６，６×１０－６Ｍ时，Ｌ型钙电流的峰值电流分别下降１８．５％、２８．８％、４５．３％，Ｐ均＜０．０５；对内向整流性钾流基本无影响。可见普罗帕酮不单纯是钠通道阻滞剂，对钙通道也有较强的阻滞作用     

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