快速心房起搏对家兔L型钙通道和钾通道Kv4.3mRNA与蛋白表达的影响

目的建立家兔快速心房起搏模型,探讨快速心房起搏早期心房超微结构变化和相关离子通道基因和蛋白表达的改变。方法36只家兔给予600次／min的频率进行心房起搏,按起搏时间分成6组,透射电镜观察心房肌超微结构变化,用反转录聚合酶链反应进行检测L型钙通道αlc、B1、以亚单位和钾通道Kv4．3的mRNA表达,Westernbolt检测L型钙通道αlc、钾通道Kv4．3的蛋白表达。结果心房肌细胞超微结构的改变在起搏3h后出现,随着起搏时间的延长,出现线粒体空泡化、肌丝溶解和糖原聚集。L型钙通道的αlc、β1的mRNA表达下调出现在起搏6h后;在24h后并没有随着起搏时间的延长而进一步下降,钾通道Kv4．3的mRNA表达在起搏24h后开始明显下降,以亚单位的表达水平在起搏后没有改变。L型钙通道的αlc、钾通道Kv4．3的蛋白表达水平改变与相应的mRNA表达改变相平行。结论在快速起搏早期,L型钙通道和钾通道Kv4．3的基因和蛋白表达水平下降,心房肌细胞超微结构改变早于离子通道表达水平发生变化,主要的机制可能是与快速起搏引起的钙超载导致离子通道转录水平的下降有关。     

快速起搏大鼠心房肌细胞L-型钙通道及钾通道Kv4.3的表达变化

目的 利用原代培养的心房肌细胞建立快速起搏模型,研究L-型钙通道及钾通道Kv4.3在快速起搏早期的表达变化.方法 原代培养大鼠心房肌细胞,并建立快速起搏细胞模型,利用RT-PCR以及Western-blot方法检测L-型钙通道α1c及钾通道Kv4.3在快速起搏3、6、12、24 h后mRNA和蛋白的表达变化.结果 快速起搏6 h后L-型钙通道α1c的mRNA和蛋白表达较起搏前持续降低,并于24 h时达到最低值;而钾通道Kv4.3 mRNA和蛋白的表达在快速起搏12 h后降低,并且在其后保持相对稳定的水平.结论 快速起搏早期,原代培养心房肌细胞L-型钙通道α1c及钾通道Kv4.3的mRNA和蛋白表达均出现不同程度的降低,提示其发生了离子通道重构,并且可能是电重构的分子基础.     

辛伐他汀对快速起搏早期家兔心房肌超微结构的影响及可能机制

目的建立家兔快速心房起搏模型,探讨辛伐他汀对快速心房起搏早期心房超微结构及L型钙通道α1c蛋白的影响。方法 30只家兔随机分为对照组、心房快速起搏组、辛伐他汀组各10只。辛伐他汀组5 mg·kg~(-1)·d~(-1)辛伐他汀灌胃2 w,其余组以等量生理盐水灌胃,对照组不行起搏,心房快速起搏组和辛伐他汀组行快速心房起搏(800次/min)建立家兔快速起搏模型,急性起搏8 h后取右房组织,电镜观察心房肌超微结构的改变,免疫组化法观察急性起搏8 h后右房组织L型钙通道α1c蛋白表达。结果心房快速起搏组起搏8 h后心房肌细胞超微结构有明显变化,如肌原纤维排列紊乱、肌小节减少,肌溶解、线粒体肿胀、空泡样改变、糖原聚集等心房结构重构的改变,心房肌细胞L型钙通道的α1c亚单位的蛋白表达水平较对照组明显下降。辛伐他汀组心房肌超微结构的改变相对较轻,L型钙通道α1c亚单位的蛋白表达水平较对照组下降不明显。结论辛伐他汀可明显改善快速起搏早期家兔心房肌超微结构的变化,对快速心房起搏导致的L型钙通道α1c亚单位的表达有一定保护作用。     

犬上腔静脉肌袖与右房心肌某些通道亚单位mRNA的表达

目的研究犬上腔静脉肌袖与右房游离壁快速激活延迟整流钾电流(IKr),L型钙电流(ICa-L),短暂外向钾电流(Ito)通道亚单位mRNA表达水平。方法8只健康杂种犬,取上腔静脉肌袖及右房游离壁,采用逆转录聚合酶链反应的方法测定IKrα亚单位ERG、ICa-Lα1亚单位CaV1.2、Itoα亚单位Kv4.3及β亚单位KChIP2mRNA表达水平并进行半定量分析。结果上腔静脉肌袖中ERG表达水平高于右房(P<0.05),而CaV1.2、Kv4.3、KChIP2的mRNA表达均低于右房(P<0.05)。结论上腔静脉肌袖与右房之间存在离子通道基因表达水平的差异。     

犬右房不同部位Kv4.3、KchIP2、Cav1.2 mRNA的表达

目的研究犬右房不同部位短暂外向钾电流、L型钙电流亚单位mRNA的表达情况,探讨其在致房性心律失常中的意义。方法应用逆转录-聚合酶链反应半定量分析犬界嵴、梳状肌、右心耳的短暂外向钾电流α亚单位(Kv4.3)、β亚单位(KchIP2)及L型钙电流的α亚单位(Cav1.2)mRNA的表达量(以β-actin为内参照)。结果界嵴和梳状肌Kv4.3、KchIP2 mRNA高于右心耳(P<0.05或0.01);界嵴Cav1.2 mRNA高于梳状肌和右心耳(P均<0.05),而梳状肌和右心耳之间没有差异。结论Kv4.3、KchIP2、Cav1.2 mRNA在右房空间表达上的差异与其相应离子流在右房空间上的差异一致,可能是其离子流差异的分子基础。     

钙离子通道基因mRNA表达在心房颤动发生机制中的作用

目的:探讨钙离子通道基因mRNA表达在心房颤动(房颤)发生机制中的作用.方法:杂种犬15只,随机分为3组,分别为正常对照组、单纯房颤组和房颤加mibefradil组.房颤组置入埋藏式高频率心脏起搏器,起搏频率520次/min.正常对照组不置入起搏器.术后观测24周.采用RT-PCR扩增心房肌组织的L型钙通道α1亚单位、T型钙通道α1H亚单位、钠/钙交换体和Kv4 3基因的mRNA.结果:单纯房颤组与正常对照组相比较,L型钙钙通道α1亚单位的mRNA有轻微下调(P>0 05),T型钙通道α1H亚单位的mRNA明显上调(P<0 05),钠/钙交换体的mRNA下调13%(P>0 05),Kv4 3基因的mRNA明显下调(P<0 05).房颤加mibefradil组与单纯房颤组相比较,钠/钙交换体的mRNA上调10%(P>0 05),Kv4 3基因的mRNA上调(P<0 05).结论:持续性房颤6个月时T型钙通道α1H亚单位mRNA的表达显著增加而Kv4 3 mRNA的表达显著下调,可能是持续性房颤钙超载的主要原因.而L型钙通道α1亚单位和钠/钙交换体mRNA的表达无明显改变,提示它们可能在房颤的发生机制中不起重要作用.     

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     

房颤心房肌L型钙通道α1c亚单位mRNA的表达及意义

目的研究房颤心房肌L型钙通道结构重塑的分子基础。方法心脏手术中采集慢性房颤及窦性心律患者的右心房肌,提取总RNA行逆转录,用PCR技术检测L型钙通道α1c亚单位不同位置的mRNA片段表达。结果①α1c亚单位Ⅰ~Ⅱ跨膜区连接对应的mRNA丰度在慢性房颤心房肌与窦性心律心房肌无明显差异。②α1c亚单位Ⅳ跨膜区对应的mRNA丰度则慢性房颤心房肌较窦性心律心房肌明显降低。结论房颤心房肌L型钙通道结构重塑与α1c亚单位的亚型表达改变有关。     

房颤心房肌L型钙通道仅α1c亚单位mRNA的表达及意义

目的研究房颤心房肌L型钙通道结构重塑的分子基础。方法心脏手术中采集慢性房颤及窦性心律患者的右心房肌，提取总RNA行逆转录，用PCR技术检测L型钙通道α1c亚单位不同位置的mRNA片段表达。结果①α1c亚单位Ⅰ～Ⅱ跨膜区连接对应的mRNA丰度在慢性房颤心房肌与窦性心律心房肌无明显差异。②α1c亚单位N跨膜区对应的mRNA丰度则慢性房颤心房肌较窦性心律心房肌明显降低。结论房颤心房肌L型钙通道结构重塑与α1c亚单位的亚型表达改变有关。     

肾性高血压大鼠心房肌电重构相关离子通道的mRNA的变化

目的利用"2肾1夹"型Goldblatt高血压大鼠模型,观察在高血压形成过程中与心房肌电重构相关的离子通道mRNA的变化,探讨其意义.方法 Sprague-Dawley大鼠,高血压组用U型银夹夹住左肾动脉近心端,右肾保留;假手术组只分离左肾动脉,不夹银夹.套尾法监测尾动脉血压,分别于手术后2、4、6 w处死动物(各6只),TRIZOL提取左右心耳总mRNA,RT-PCR半定量分析Na+通道α亚单位(Na+-α)、L型钙通道α1C亚单位(CaL-α1C)和瞬时外向钾通道(Kv4.3)mRNA的表达量(以GAPDH为内参照).结果在高血压形成过程中,左心耳CaL-α1C的mRNA水平在2 w和4 w明显增高,分别是假手术组的2.5倍和2.4倍(P<0.001),6 w恢复正常,右心耳在2 w和4 w无明显变化,6 w为假手术组的2.5倍(P<0.001);左心耳Kv 4.3的mRNA水平在4 w和6 w分别是假手术组的1.9倍和1.7倍(P<0.001),在右心耳却呈下降趋势,4 w和6 w分别降低了30%(P<0.05)和20%(P=0.10);Na+-α的mRNA在左右房无明显变化.结论肾性高血压大鼠在高血压形成过程中,心房肌CaL-α1C的mRNA水平表现为上调,左房明显早于右房,左右房Na-α和Kv 4.3的mRNA变化明显不平行;提示mRNA水平的变化是电重构的基础,并可能进一步导致心律失常的发生.     

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.     

Phosphoinositide isoforms determine compartment-specific ion channel activity

Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments, and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is a plasma-membrane (PM)-specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly localized channels are similarly "inactivated" at the PM, we studied PIP(2) modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch-clamping isolated lysosomes, we previously found that lysosomal, but not PM-localized, TRPML1 is active with PI(3,5)P(2), a lysosome-specific PIP(2), as the underlying positive cofactor. Here we found that "silent" PM-localized TRPML1 could be activated by depleting PI(4,5)P(2) levels and/or by adding PI(3,5)P(2) to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P(2). Conversely, depletion of PI(4,5)P(2) by either depolarization-induced activation or chemically induced translocation of 5'-phosphatase potentiated whole-cell TRPML1 currents. PI(3,5)P(2) activation and PI(4,5)P(2) inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP(2)-interacting domain. Thus, PI(4,5)P(2) may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters.     

Leptin-stimulated KATP channel trafficking

Insulin secretion from pancreatic β-cells is initiated by the closure of ATP-sensitive K⁺ channels (K_ATP) in response to high concentrations of glucose, and this action of glucose is counteracted by the hormone leptin, an adipokine that signals through the Ob-R_b receptor to increase K_ATP channel activity. Despite intensive investigations, the molecular basis for K_ATP channel regulation remains uncertain, particularly from the standpoint of whether fluctuations in plasma membrane K_ATP channel content underlie alterations of K_ATP channel activity in response to glucose or leptin. Surprisingly, newly published findings reveal that leptin stimulates AMP-activated protein kinase (AMPK) in order to promote trafficking of K_ATP channels from cytosolic vesicles to the plasma membrane of β-cells. This action of leptin is mimicked by low concentrations of glucose that also activate AMPK and that inhibit insulin secretion. Thus, a new paradigm for β-cell stimulus-secretion coupling is suggested in which leptin exerts a tonic inhibitory effect on β-cell excitability by virtue of its ability to increase plasma membrane K_ATP channel density and whole-cell K_ATP channel current. One important issue that remains unresolved is whether high concentrations of glucose suppress AMPK activity in order to shift the balance of membrane cycling so that K_ATP channel endocytosis predominates over vesicular K_ATP channel insertion into the plasma membrane. If so, high concentrations of glucose might transiently reduce K_ATP channel density/current, thereby favoring β-cell depolarization and insulin secretion. Such an AMPK-dependent action of glucose would complement its established ability to generate an increase of ATP/ADP concentration ratio that directly closes K_ATP channels in the plasma membrane.     

心血管离子通道病的研究现状

心血管离子通道病是离子通道病的重要组成部分。作为原发或继发病因离子通道病变几乎参与了所有的心血管疾病的发生发展过程，是心脏性猝死的主要原因。本文对遗传性心脏离子通道病、获得性心脏离子通道病以及离子通道与血管疾病进行综述，并简要介绍了心血管离子通道病的治疗。     

气道黏液高分泌与上皮通道蛋白关系的研究进展

COPD、哮喘、支气管扩张及肺囊性纤维化等气道高分泌疾病中的分泌物潴留除了与黏蛋白的绝对量增多有关,还与黏蛋白／水盐比例失衡密切相关。气道上皮细胞的各种通道蛋白如Na^＋、Cl^-和水通道等参与气道表面液体中水盐跨膜转运,通过调节黏蛋白／水盐比例来影响黏液的总量和黏滞度,在抗黏液高分泌中显示出广泛的前景。     

Electrophysiologic effects of potassium channel openers

Potassium-channel openers or activators have been introduced as a new class of antihypertensive and antianginal agents that act by increasing membrane conductance to potassium, mainly through augmentation of the ATP-sensitive potassium current. Recent in vitro studies have shown that K⁺-channel openers exert concentration-dependent effects on cardiac electrophysiology. A shortening of the cardiac action potential by acceleration of repolarization has been reported in multicellular preparations as well as in isolated myocytes. However, drug concentrations that affect the action potential duration of myocardial cells are considerably higher (10- to 100-fold) than those needed for effects on vascular smooth muscle cells. Studies in which mostly high concentrations of K⁺-channel openers were used have demonstrated that these drugs may accelerate automaticity and may promote reentrant activity. Particular interest has focused on the question whether opening of potassium channels may be potentially arrhythmogenic in the setting of acute myocardial ischemia. On the other hand, recent studies have shown that K⁺-channel openers are effective in suppressing polymorphic ventricular tachyarrhythmias induced by early afterdepolarizations and triggered activity in vivo. The clinical relevance of these experimental studies to the clinical situation is still unclear. Some K⁺-channel openers have been shown to produce electrocardiographic T-wave changes in patients in whom their effectiveness as antihypertensives was tested. However, this effect was not associated with adverse effects and has not been demonstrated for all compounds. So far the worsening of existing arrhythmias or the induction of new arrhythmias by K⁺-channel openers in humans has not been reported.     

Molecular basis of multistep voltage activation in plant two-pore channel 1

Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge–bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca²⁺ and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.

Voltage-gated ion channels (VGICs) use voltage-sensing domains (VSDs) to sense changes in electrical potential across biological membranes (1, 2). VSDs are composed of a four-helix bundle, in which one helix carries charged residues that move in response to changes in transmembrane electric field (3, 4). VSDs usually adopt a “resting state” when the membrane is at “resting potential”: ∼ −80 mV for animal and ∼ −150 mV for plant plasma membranes (5). In comparison, much lower resting membrane voltages are set for intracellular endo-membranes: ∼ −30 mV across the plant vacuole (6) and mammalian lysosome (7). As the membrane potential vanishes during depolarization, so does the downward electrostatic force on the cationic side chains causing them to relax toward the outside of the membrane across a hydrophobic constriction site (HCS) or hydrophobic seal (8). This conformational change is conveyed to the central pore, formed by four pore domains in a quasi-fourfold arrangement, which dilates to allow the diffusion of ions down their electrochemical gradients. The exact nature of the conformational change in VSDs has been the subject of decades of biophysical investigation (9–12), though to this date, only a few structural examples exist of voltage sensors in resting or multiple conformations (13–18).Two-pore channels (TPCs) are defined by their two tandem Shaker-like cassette subunits in a single polypeptide chain, which dimerize to form a C2-symmetric channel with four subunits and 24 (4 × 6) transmembrane helices (19–21). There are three TPC channels, TPC1, 2, and 3, each with different voltage or ligand gating and ion selectivity. Among the voltage-gated TPCs (all except lipid-gated TPC2), only the second VSD (VSD2) is electrically active (18, 22–24), while VSD1 is insensitive to voltage changes and is likely static under all changes in potential.In plants, the vacuole comprises up to 90% of the plant cell volume and provides for a dynamic storage organelle that, in addition to metabolites, is a repository for ions including Ca²⁺. TPC1 channels confer excitability to this intracellular organelle (25) and, unlike other TPCs, are calcium regulated: external Ca²⁺ (in the vacuolar lumen) inhibits the channel by binding to multiple luminal sites, while cytosolic Ca²⁺ is required to open the channel by binding to EF hands, although the exact mechanism by which this activation occurs is unknown (22). These electrical properties allowed our group and Youxing Jiang’s group to determine the first structure of an electrically resting VGIC by cocrystallizing the channel with 1 mM Ca²⁺, which maintains the VSD in a resting configuration at 0 mV potential (16, 22).Previously (17), we used a gain-of-function mutant of AtTPC1 with three luminal Ca²⁺-binding acidic residues on VSD2 neutralized (D240N/D454N/E528N) termed AtTPC1_DDE (abbreviated here as DDE) to visualize channel activation at the level of atomic structure, but we were unable to sufficiently resolve details of the electrically active VSD2 due to structural heterogeneity. In addition, the intracellular activation gate remained closed. We now present multiple structures of intermediately activated states of AtTPC1 determined by extensive image processing. In order to visualize such states, we modulated the channel’s luminal Ca²⁺ sensitivity using a well-studied gain-of-function single-point mutant, D454N (fou2), and also the triple mutant DDE for comparison. fou2 is known to desensitize the channel to inhibitory, external (luminal) calcium ions (26, 27). Mutations in D454 and closely related luminal Ca²⁺-binding carboxyls to alanine D240A, D454A, E528A (termed AtTPC1ΔCa_i) were previously shown to effectively attenuate the Ca²⁺-induced shift of the voltage activation threshold of AtTPC1 to depolarizing potentials at high luminal Ca²⁺ (28).The fou2 and DDE mutations lie in the coordination sphere of the inhibitory Ca²⁺ site on the luminal side of the VSD2–pore interface formed by D454, D240, and E528. The D454N mutation in the fou2 channel enhances the defense capacity of plants against fungal or herbivore attack due to increased production of the wounding hormone jasmonate (29). These effects on plant performance and defense are probably due to short circuiting of the vacuolar membrane (26, 27, 30) in which TPC1 has increased open probability at resting potential. Compared to wild-type (WT) TPC1, the activation threshold in the fou2 channel is shifted to more negatively polarized potentials and also has significantly lower sensitivity to inhibitory Ca²⁺ in addition to exhibiting faster activation kinetics than its WT counterpart that was originally named the “slow vacuolar” (SV) channel due to its slow conductance onset (20, 21, 30). Therefore, D454N confers more than just reduced sensitivity to external Ca²⁺ but intrinsic hyperactivity as well. Our structures of these AtTPC1 mutants attempt to explain how the voltage sensor functions during electrical activation and how exactly luminal Ca²⁺ affects this process.     

钠离子通道与慢性心力衰竭

钠离子通道能产生对心肌细胞动作电位发生和传播起重要作用的快钠电流,还能产生影响动作电位时程的晚钠电流（INaL）。钠离子通道功能的改变是多种心血管疾病的发病基础,心血管疾病发生后也会产生钠离子通道重构。慢性心力衰竭（HF）是临床常见的心血管病综合征,钠离子通道,特别是INaL与慢性HF的研究已成为近期的热点,本文就钠离子通道与慢性HF的相互关系进行了综述。     

Voltage-sensor movements in the Eag Kv channel under an applied electric field

Voltage-dependent ion channels regulate the opening of their pores by sensing the membrane voltage. This process underlies the propagation of action potentials and other forms of electrical activity in cells. The voltage dependence of these channels is governed by the transmembrane displacement of the positive charged S4 helix within their voltage-sensor domains. We use cryo-electron microscopy to visualize this movement in the mammalian Eag voltage-dependent potassium channel in lipid membrane vesicles with a voltage difference across the membrane. Multiple structural configurations show that the applied electric field displaces S4 toward the cytoplasm by two helical turns, resulting in an extended interfacial helix near the inner membrane leaflet. The position of S4 in this down conformation is sterically incompatible with an open pore, thus explaining how movement of the voltage sensor at hyperpolarizing membrane voltages locks the pore shut in this kind of voltage-dependent K⁺ (K_v) channel. The structures solved in lipid bilayer vesicles detail the intricate interplay between K_v channels and membranes, from showing how arginines are stabilized deep within the membrane and near phospholipid headgroups, to demonstrating how the channel reshapes the inner leaflet of the membrane itself.

In voltage-dependent ion channels, the transmembrane voltage determines whether the pore opens. At the same time, the flow of ions through the open pore alters the membrane voltage by charging the membrane capacitance. This recursive regulation of ion channel activity by membrane voltage is the fundamental process at the heart of cellular electricity. As described by Hodgkin and Huxley (1), voltage-dependent membrane permeability to Na⁺ and K⁺ (ion channels as molecular entities had not yet been discovered) generates the action potential, which is by far the most rapid form of information transfer across long distances in cells. Voltage-dependent ion channels underlie many other aspects of cell signaling as well, including the initiation of muscle contraction by voltage-dependent Ca²⁺ channels (2, 3) and the control of cardiac and neuronal pacemaker frequency by the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel (4, 5).Voltage-dependent ion channels contain structural domains called voltage sensors that control pore opening by membrane voltage. Whether in K⁺, Na⁺, Ca²⁺, or cation channels like HCN channels or transient receptor potential (TRP) channels, voltage sensors have a conserved structure comprising four transmembrane helices, named S1, S2, S3, and S4 (3, 6, 7). The fourth helix, S4, contains repeats of the amino acid triplet (RXX)_n, where R stands for arginine, sometimes substituted by lysine, X for hydrophobic amino acid, and n varies widely among different channels. At the center of voltage sensors, inside the membrane’s interior, a constellation of negative charged amino acids, aspartate or glutamate, and a phenylalanine residue forms a gating-charge transfer center that stabilizes the positive charged side chains of arginine and lysine as they cross the membrane (8, 9). In some voltage sensors, S4 undergoes a transition from an α to a 3₁₀ helix to direct the arginine and lysine side chains of S4 into the gating-charge transfer center (8–11). The displacement of S4 across the membrane is detectable as a nonlinear capacitive current, called gating current in electrophysiology experiments (9, 12), and is ultimately responsible for voltage control of a voltage-dependent ion channel’s pore.So far, voltage-dependent ion channel structures have been determined in crystals, detergent micelles, or nanodiscs without a voltage difference across them (8, 13–19). Under such conditions, most voltage sensors adopt a depolarized conformation, which is expected in a membrane at 0 mV. Chemical cross links, metal affinity bridges, mutations, and toxins have been used to capture or stabilize voltage sensors in conformations thought to mimic the hyperpolarized (i.e., negative voltage inside) condition (10, 11, 20–23). Here, we present a cryo-electron microscopy (cryo-EM) analysis of the mammalian Eag voltage-dependent K⁺ (K_v) channel in lipid membrane vesicles with a voltage generated across the membrane using K⁺ ion gradients in the presence of valinomycin. Doing so allows us to not only visualize how the voltage sensors respond to the applied electric field but also to see how the lipid membrane near the channel, which is intimately tied to the function of voltage-dependent ion channels, is reshaped by these conformational changes.     

心房颤动患者乙酰胆碱依赖性钾通道和快速延迟整流钾通道基因表达的研究

目的研究心房颤动(AF)患者心房组织中乙酰胆碱依赖性钾通道(Kach)和快速延迟整流钾通道(Kr)的基因表达。探讨AF时心房组织Kach和Kr的mRNA表达改变及其意义。方法自20例因风湿性心脏病或先天性心脏病接受外科手术的患者,于术中采取的右心耳标本分为2组,窦性心律(SR)组14例,慢性AF组6例,应用半定量逆转录聚合酶链反应(RTPCR)技术,检测心房组织Kach和Kr基因表达。结果心房组织中Kir34(Kach的一种成分)和KrmRNA的表达,与SR组051±005相比AF组029±005下调43%,差异有显著性(P<0001),而KrmRNA水平在SR组056±003和AF组057±003之间无明显改变,差异无显著性(P>005)。结论房颤患者KachmRNA表达下调,可能是心房肌细胞IKach密度减弱的分子基础。Kr转录水平无明显改变。