FHL2蛋白及其参与心脏离子通道电流调控的研究进展

FHL蛋白是仅由4个半uM结构域和位于N末端的一个锌指结构组成的一类蛋白。由于这一类蛋白缺少其他功能性或结构性的结构域,因而被归为LIM—only蛋白家族。FHL2属于FHL家族。人类的FHL家族包括FHL1、FHL2、FHL3、FHT4和ACT。FHL2参与了多种蛋白质的相互作用,同时也参与了多种骨架蛋白、酶、转录因子等的调控作用。新近的研究发现,FHL2与一些心脏离子通道存在相互作用并对这些通道进行功能调控。本文就FHL2及其对离子通道的调控作用研究进展做一综述。     

FHL2在消化系统恶性肿瘤中的研究进展

FHL2是一种参与信号转导和基因转录的支架蛋白,具有FHL蛋白典型的结构特征. FHL2含有四个半LIM结构域,不同的LIM结构域可以与不同的蛋白质结合,从而激活或者抑制转录因子如P53、血清应答因子等的活性,进而影响肿瘤的发生发展.既往研究发现, FHL2在肿瘤发生发展中具有复杂的生物学作用,在不同类型肿瘤中发挥促癌或抑癌的作用.本文就FHL2在消化系统恶性肿瘤中的研究进展作一概述.     

与HERG钾通道相互作用的FHL2蛋白对该通道功能的调控

目的 筛选与心脏人类果蝇相关基因（HERG）的编码蛋白钾通道存在相互作用的蛋白质,并进一步研究该相互作用蛋白对HERG钾通道的调控作用。方法 ①以带有编码人类心脏HERG钾通道的cDNA为模板,通过PCR方法得到编码人类心脏HERG钾通道氨基末端（404个氨基酸）的DNA片段。将该片段克隆入pGBKT7载体, 构建“诱饵”质粒pGBKT7-HERG-NT。②应用酵母双杂交技术筛选人类心脏cDNA文库。③以PCR法扩增4个半LIM结构域（FHL2）基因的开放读框片段(ORF),并克隆入pcDNA3.0载体。④以pcDNA3.0-herg转染HEK293细胞,应用G418筛选得到HEK293/HERG细胞株。以pcDNA3.0-FHL2转染HEK293细胞,筛选得到HEK293/FHL2细胞株后,再将pcDNA3.0-herg转染入该细胞株。⑤应用膜片钳技术,研究FHL2对HERG通道功能的影响。结果 ①用酵母双杂交技术筛选得到37个阳性克隆,其中含有表达FHL2蛋白的克隆。②膜片钳检测发现,FHL2蛋白在增加HERG电流幅度的同时并调节其激活过程。 结论 FHL2蛋白能与HERG氨基末端相互作用而影响HERG钾通道的功能。     

酪氨酸的磷酸化及去磷酸化对心脏钾离子通道的调控

心脏钾离子通道是人体内广泛存在的、种类最多、作用最复杂的一类离子通道,并在人类心肌细胞动作电位的各个时程中发挥着重要的作用。近年来由于新兴技术如膜片钳技术、基因突变技术、分子克隆技术等诸多技术的运用和发展,人们对钾离子通道的基因表达、分子结构、生理病理作用及调控机制等方面都有了很深的认识,然而对蛋白磷酸化与去磷酸化对心脏钾离子通道的调控尤其是酪氨酸磷酸化和去磷酸化调控方面的研究却比较少,为此做一简单综述。     

疟原虫有性阶段转录调控的研究进展

疟疾是由疟原虫感染所致的传染性疾病。疟疾的传播依赖于疟原虫在脊椎动物和按蚊两个宿主内的交替发育。疟原虫有性配子体阶段是其从脊椎动物传递至按蚊的唯一阶段。疟原虫的有性转化、有性发育和配子发生对疟原虫的传播起重要作用。深入了解疟原虫有性阶段的基因表达及相关调控机制,有助于筛选新的抗疟药物或疫苗靶点。本文对疟原虫有性阶段基因表达转录调控的研究进展进行综述,以期为阻断疟疾传播提供参考。     

GST—FHL2融合蛋白的表达及纯化

目的高效表达和纯化可溶性GST—FHL2融合蛋白。方法（1）PCR法扩增FHL2（Four and a half LIM domains2）基因的编码片段，分别在5′端和3′端加上EcoR Ⅰ和Xho Ⅰ酶切位点，并克隆进入原核表达载体pGEX-4T-1；（2）利用异丙基硫代-β-D-半乳糖苷（IPTG）诱导重组质粒pGEX4T-1-FHL2在大肠杆菌B121（DE3）中表达同时带有谷胱甘肽-S-转移酶（GST）标签的融合蛋白；（3）超声法裂解大肠杆菌，应用谷胱苷肽琼脂糖树脂纯化可溶的GST—FHL2融合蛋白；（4）通过SDS—PAGE和Western blot验证GST—FHL2的表达。结果（1）成功构建pGEX-4T-1-FHL2重组质粒，测序结果证明FHL2与载体的GST在同一读框；（2）0．1mmol／L的IPTG在23℃的条件下能诱导可溶性GST—FHL2融合蛋白高效表达；（3）在Western blot分析中，GST—FHL2能被鼠抗GST单克隆抗体特异性识别，条带所在位置和GST-FHL2的分子量相符。结论正确构建pGEX-4T-1-FHL2重组质粒，在大肠杆菌BL21中高效表达GST—FHL2融合蛋白，经谷胱苷肽琼脂糖树脂纯化得到高纯度的可溶性GST-FHL2融合蛋白。     

钾离子通道与肥胖致心律失常的研究进展

肥胖易诱发心律失常，并且治疗手段有限。现有证据表明肥胖致心律失常与心脏钾离子通道异常有关。研究肥胖患者心脏钾离子通道功能表达和电生理机制，有利于提高对肥胖致心律失常的认识，发现新的致病机制，找出新的治疗靶点，对于肥胖患者意义重大。本文旨在探讨心脏钾离子通道与肥胖致心律失常的研究进展。     

应用酵母双杂交技术筛选转录因子MEOX2的相互作用蛋白

目的研究同源盒基因Meox2所编码的转录因子MEOX2在心肌细胞内与哪些蛋白质存在相互作用。方法应用酵母双杂交技术对人心脏cDNA文库进行筛选。（1）通过PCR方法扩增编码组氨酸／谷氨酸（H／Q）富含区缺失的MEOX2蛋白质（Meox2△HQ）的DNA片断，并克隆进入pGBKT7载体，从而构建“诱饵”质粒pGBKT7-Meox2△HQ；（2）将“诱饵”质粒pGBKT7-Meox2△HQ转化酵母菌AH109，然后从被转化的酵母菌中提取蛋白质，应用抗C-Myc单克隆抗体进行免疫印迹分析，检测“诱饵”蛋白的表达；（3）将被“诱饵”质粒转化的AH109分别涂布于SD／-Trp、SD／-Trp／-His、SD／-Trp／-Ade和SD／-Trp／X-α-GAL平板上，以观察“诱饵”蛋白自我激活报告基因与否；（4）将已被“诱饵”质粒转化的AH109与人心脏cDNA文库进行杂交，筛选阳性克隆，分离阳性克隆中的cDNA，并测序。结果Meox2△HQ与pGBKT7中的Gal4 DNA结合域及C-myc在同一读框，并稳定表达融合蛋白“Gal4 DNA结合域．C-myc-Meox2△HQ”，“诱饵”蛋白不会自我激活报告基因，筛选得到11个准阳性克隆。结论同源盒基因Meox2所编码的转录因子MEOX2可能与心肌细胞中的多种蛋白质存在相互作用，并参与这些蛋白质表达的调控。     

胃癌细胞中环氧合酶-2对延迟整流性钾通道调控机制的研究

目的 探讨胃癌细胞中环氧合酶(COX)-2对延迟整流性钾通道(HERG)电流的影响和相应的调控机制.方法 ①采用逆转录聚合酶链反应(PCR)、Western印迹和膜片钳技术检测环氧合酶(COX)-2反义载体转染胃癌细胞前后HERG mRNA、蛋白和电流的变化.②采用酶联免疫吸附试验检测COX-2反义载体转染胃癌细胞前后环磷酸腺苷(cAMP)水平的变化.③采用PCR技术构建缺失cAMP结合结构域的HERG突变体,并转染胃癌细胞.④应用COX-2抑制剂和前列腺素(PG)E2作用于胃癌细胞和HERG突变体转染的胃癌细胞,观察HERG电流的变化.⑤将cAMP的拟似剂和拮抗剂、蛋白激酶(PK)A抑制剂分别作用于胃癌细胞和HERG突变体转染的胃癌细胞,观察相应的HERG电流变化.结果 ①与亲本细胞相比,COX-2反义转染胃癌细胞的HERG mRNA和蛋白表达无变化,而HERG电流强度减弱(P<0.05).②与亲本细胞相比,COX-2反义转染胃癌细胞中的cAMP水平明显下降(P<0.05).③COX-2抑制剂减弱而PGE2增强HERG电流的强度.但在缺失cAMP结合域的HERG突变体转染后的胃癌细胞,COX-2抑制剂和PGE2对HERG电流未产生明显的影响.④cAMP拟似剂可增强SGC7901细胞的HERG电流,而cAMP拮抗剂则减弱其电流.但对于缺失cAMP结合结构域的突变体转染的胃癌细胞中的HERG电流,cAMP的拟似剂和拮抗剂均未显示出明显的增强或抑制作用.⑤PKA抑制剂对SGC7901细胞和突变体转染的胃癌细胞的HERG电流均无明显影响.结论 COX-2通过其代谢产物PGE2而影响HERG电流.PGE2通过与受体结合后影响cAMP浓度,而cAMP通过与HERG蛋白上的特殊结构域结合对HERG电流产生影响,此过程不受PKA的调控.     

KCNE对心肌细胞钾离子通道的调控及与遗传性心律失常

KCNE基因家族编码蛋白MinK和MinK相关肽1-4,作为辅助性β亚基与多种钾离子通道蛋白α亚基组成电压依赖性钾离子通道复合体。KCNE家族对钾离子通道的调控作用错综复杂,对维持正常的心脏功能十分重要,其基因结构和功能的异常可导致心脏电活动紊乱,引起各种心律失常。     

Pharmacology of cardiac potassium channels

Cardiac K+ channels are membrane-spanning proteins that allow the passive movement of K+ ions across the cell membrane along its electrochemical gradient. They regulate the resting membrane potential, the frequency of pacemaker cells and the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters and hormones and class III antiarrhythmic drugs that prolong the action potential duration (APD) and refractoriness and have been found effective to prevent/suppress cardiac arrhythmias. In the human heart, K+ channels include voltage-gated channels, such as the rapidly activating and inactivating transient outward current (Ito1), the ultrarapid (IKur), rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward rectifier current (IK1), the ligand-gated channels, including the adenosine triphosphate-sensitive (IKATP) and the acetylcholine-activated (IKAch) currents and the leak channels. Changes in the expression of K+ channels explain the regional variations in the morphology and duration of the cardiac action potential among different cardiac regions and are influenced by heart rate, intracellular signalling pathways, drugs and cardiovascular disorders. A progressive number of cardiac and noncardiac drugs block cardiac K+ channels and can cause a marked prolongation of the action potential duration (i.e. an acquired long QT syndrome, LQTS) and a distinct polymorphic ventricular tachycardia termed torsades de pointes. In addition, mutations in the genes encoding IKr (KCNH2/KCNE2) and IKs (KCNQ1/KCNE1) channels have been identified in some types of the congenital long QT syndrome. This review concentrates on the function, molecular determinants, regulation and, particularly, on the mechanism of action of drugs modulating the K+ channels present in the sarcolemma of human cardiac myocytes that contribute to the different phases of the cardiac action potential under physiological and pathological conditions.     

SNARE protein regulation of cardiac potassium channels and atrial natriuretic factor secretion

Coordinated cardiac ion channel gating is fundamental for generation of action potential and excitability throughout the myocardium. The interaction of pore-forming ion channels with auxiliary subunits can regulate surface expression, localization and anchoring of these channels to plasma membrane. SNARE (soluble N-ethylmaleimide sensitive factors attachment protein or SNAP receptor) proteins mediate the targeting, docking, and fusion of intracellular vesicles for exocytotic release of neurotransmitters and hormones. In secretory neurons and neuroendocrine cells, some voltage-gated channels are physically coupled with SNARE proteins, resulting in alterations in channel gating and trafficking. Coupling of SNARE proteins to membrane ion channels is however not unique to secretory cells. We have demonstrated the expression of SNARE proteins in rodent myocardial tissue, and more importantly, functional interaction of SNARE proteins with cardiac K_ATP and K_v (K_v1.2, K_v2.1, K_v4.2, K_v4.3, and K_v11.1) channels. SNARE proteins, therefore, have similar fundamental functions in ion channel trafficking and regulation per se, independent of secretion. We now review the body of work of SNARE protein regulation on membrane ion channels in the heart.     

G-protein control of cardiac potassium channels

Two cardiac potassium (K(+)) channels are activated by pertussis toxin (PTX)-sensitive G proteins either directly or in a "membrane-delimited" manner. They are muscarinic K(+)(K(ACH)) and ATP-sensitive K(+)(K(ATP)) channels. K(ACH) channels are responsible for acetylcholine (ACh)- or adenosine-induced deceleration of the heartbeat and atrioventricular conduction, while K(ATP) channels are responsible for the ischemia-induced shortening of the cardiac action potential and possibly for the adenosine-mediated protection from ischemic damage. Distinct molecular mechanisms underlie G-protein activation of these cardiac K(+) channels; the α subunit of PTX-sensitive G proteins activates the K(ATP) channels, while βγ subunits activate the K(ACh) channel. The physiologic significance of this heterogeneous mechanism remains to be determined.     

Amiodarone inhibits cardiac ATP-sensitive potassium channels

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.     

钙调神经磷酸酶对心肌细胞离子通道的调节

钙调神经磷酸酶（calcineurin，CaN），是迄今所知的惟一一种受Ca^2＋／钙调蛋白（calmodulin，CaM）调节的丝氨酸／苏氨酸蛋白磷酸酶。CaN广泛分布于机体内各种组织中，新近研究表明CaN介导的信号通路在心血管系统的病理生理变化中发挥着重要的调节作用，如参与心肌肥大、血管平滑肌细胞增殖以及心肌离子通道功能的调节等。目前CaN在心血管系统中的作用也越来越引起人们的关注，本文就CaN的结构和功能以及其对心肌离子通道的调节作用做一概述。     

Structure and function of cardiac potassium channels.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.     

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming α-subunits. However, the molecular mechanism(s) by which phosphorylation of the α-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single α-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.