【作者】 章茜；

【导师】 董子明； Nipavan Chiamvimonvat；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 研究背景及目的房室结（atrioventricular node，AVN）是位于右心房和心室之间高度特化的传导组织，是唯一连接心房和心室之间的传导通道，也是兴奋由心房进入心室的唯一电信号通道。它的重要功能是保证心房和心室在不同的时间内有序的收缩。房室结的传导速度很慢，其中，以结区的传导速度为最慢。这一慢的传导将导致心房和心室之间的收缩延迟，以保证心室不会与心房发生同步收缩。在某些病理情况下，房室结具有进一步的功能，例如，窦房结起搏点或心房传导障碍，房室结将以它固有的自律性成为心室工作的起搏点。尽管，已知房室结的重要功能和意义，但对其电生理机理仍不明确。已有研究表明某些离子通道及离子通道基因表达在心肌细胞，包括自律细胞，例如，心脏起搏组织窦房结和房室结存在小电导-Ca²⁺-激活K⁺通道（small-conductance calcium-activated potassium channel，SK channel）。SK通道是K⁺选择性，非电压依赖性，唯一通过细胞内Ca²⁺激活的一种钾通道。在哺乳动物脑内，SK通道主要有3种亚型：SK1、SK2和SK3通道，它们对蜂毒明肽（apamin）-SK通道的阻断剂的敏感性不同，其中，SK2通道对apamin最敏感，其次是SK3通道，SK1对apamin的敏感性较低。迄今为止，已编码至少3个明确的SK通道基因，即KCNN1（SK1）、KCNN2（SK2）和KCNN3（SK3）。SK通道广泛存在于机体不同的组织，包括脑、周围神经、肝脏和平滑肌。SK通道在心脏表达的功能意义，鲜为人知。Dr．Chiamvimonvat实验室近来证明人和小鼠心肌细胞存在SK2通道，明显参与动作电位的复极化过程，且在心房的表达明显高于心室。进一步研究发现，过表达SK2通道的小鼠易发生心律失常，并伴有明显的房室结功能及房-室传导异常。本研究采用SK2通道过表达小鼠和SK2通道敲除小鼠，分别检测了它们对房室结功能的影响，首次证明了SK2通道在房室结的重要功能意义。通过建立HEK（Human embryonic kidney）293细胞SK2通道表达细胞株，探讨了SK2通道的调节及其与α-actinin2（肌动蛋白细胞骨架的结合蛋白）相互作用的分子机理。方法1．本研究采用16-22w，体重20-25g，具有C5781／6J背景的SK2通道过表达小鼠（SK2+／T），SK2通道敲除小鼠（SK2+／A，SK2A／A）及SK2野生型小鼠（Wild-type，WT），雌雄不拒。2．动物心电图（Electrocardiogram，ECG）记录：上述3组动物，腹腔注射苯巴比妥钠麻醉（60mg／kg），37℃条件下行肢体导联记录。3．AVN自发性动作电位（action potential，AP）记录：动物麻醉同上，取出心脏，暴露右心房，分离房室结区。采用3mol／L KCl微电极细胞内记录SK2+／T、SK2+／△及WT组房室结自发性AP。4．AVN细胞分离及全细胞IK，ca的记录：WT、SK2+／T and SK2+／△小鼠AVN单个细胞的分离参照以往的方法并进行改良。单个AVN细胞Ca²⁺激活K⁺电流(Ca²⁺-activited K⁺ current，IK，ca)的记录采用全细胞膜片钳法，钳制电压为-55mV，给予阶梯去极化脉冲刺激从-90mV～+40mV，滤波2KHz，采样频率10KHz。记录给予apamin（500pmol／L）前后的IK，ca，室温下进行。Apamin-敏感K⁺电流的计算为apamin（500pmol／L）给药前后Ik，ca电流密度的差（值）。5．双重免疫组化荧光标记：取WT和SK2△／△小鼠新鲜分离的心肌细胞，4％多聚甲醛固定，1％牛血清白蛋白封闭，加入一抗，4℃过夜。双重免疫组化荧光标记分为2组①抗SK2抗体（1∶100稀释）+α-actinin2抗体（1∶800）；②抗SK2抗体（1∶100）+抗NF 160KD（neurofilament 160 KD，NF160 KD）抗体（1∶100）。二抗为FITC-羊抗兔IgG（1∶250）和TEXRED-羊抗鼠IgG（1∶250）。以二抗作为阴性对照，同时进行。6．免疫组织化学：制备WT及SK2基因敲除小鼠（SK2A／A）心脏石蜡系列切片，各取WT及SK2△／△相邻心脏切片，5％羊血清封闭，抗SK2抗体（1∶200）与切片孵育，4℃过夜，再与生物素标记的二抗反应。7．建立SK2通道表达的HEK293细胞株：HEK293细胞株在Dulbecco’s modified Eagle’s培养液常规条件下培养并传代（37℃in an air／5％CO2）。参照Lipofectamine^{T M}2000试剂盒说明书，下列质粒分别转染入HEK293细胞：1)pSK2-IRES-EGFP&pcDNA3-α-actinin2；2)pSK2-IRES-EGFP。8．膜片钳记录：转染后36～48 hrs的HEK293细胞在室温条件下行全细胞膜片钳Ik，ca记录。记录前4hrs，将已转染SK2+和α-actinin2质粒的细胞给予Cytochalasin D（CyD，2.5μmol／L）。Ik，ca记录程序和条件同上述AVN细胞。9．HEK293细胞SK2和α-actinin2蛋白的表达：Western blot检测共转染α-actinin2和SK2的HEK293细胞α-actinin2和SK2蛋白的表达。10．免疫组化荧光标记：将共转染SK2+α-actinin2质粒后2-3 day的细胞2％多聚甲醛固定，1％牛血清白蛋白封闭，加入抗SK2抗体（1∶100）+抗α-actinin2抗体（1∶800），4℃过夜。二抗为FITC-羊抗兔IgG（1∶250）和TEXRED-羊抗鼠IgG（1∶250）。结果1．SK2+／T和SK2+／△小鼠显示窦房结和AVN功能异常：心电图的记录表明SK2+／△小鼠与WT比较，P-R间期明显延长，并伴有窦性心动过缓。相反，SK2+／T小鼠R-R间期及P-R间期明显缩短。提示过表达SK2基因和SK2基因敲除小鼠存在窦房结和房室结起搏功能和房-室传导功能的明显异常。2．SK2+／T小鼠AVN着火率增加，相反的结果见SK2+／△小鼠：分离的AVN自发性AP具有慢的4期自动去极化，且除极速率慢，无明显超射。这些特征表明其自发性AP来自AVN。与WT比较，SK2+／T组AVN自发性AP的频率增加，而SK2+／△小鼠AVN着火频率明显减小。详细分析自发性AP发现其周期长度（cycle-length，CL）、动作电位持续时间（action potential duration，APD）及舒张期自动去极化速度（the rate of diastolic depolarization，DDR）有明显变化。与WT比较，过表达SK2通道引起AP复极达80％（APD80）的时间缩短，DDR增快及相应的CL缩短。相反的结果见于SK2通道缺失小鼠。结果表明，SK2通道表达异常可明显影响AVN细胞自发性着火频率，进而影响心脏房-室传导。3．单个AVN细胞IK，Ca的变化：电流密度-电压关系曲线证明这一电流具有内向整流的特征，通道电流的大小随电压升高而减低，亦与时间无关，其电流的外向及内向成份都可被apamin所阻断。反转电位约-80 mV，符合Nernst方程。SK2+／T小鼠，其AVN细胞apamin敏感K⁺电流明显大于WT；而SK2通道敲除小鼠，其apamin敏感K⁺电流明显小于WT。结果表明房室结细胞存在apamin敏感的Ik，ca。推测其Ik，ca的变化是AVN细胞自发性AP改变的直接原因。4．激光共聚焦显微镜：单个AVN细胞具有对特异性SK2抗体的免疫阳性反应。心房和心室细胞也具有SK2蛋白的阳性信号。NF160作为心脏起搏组织和传导系统的标志，其阳性反应仅见于AVN细胞，以区别心室或心房细胞。5．AVN SK2免疫阳性反应：心脏组织切片AVN，心房及室间隔均可见SK2蛋白阳性反应（棕色）。与WT比较，无明显阳性反应呈现于SK2△／△小鼠心肌组织，包括AVN。结果表明SK2免疫阳性反应存在于房室结。采用H&E和Trichrome染色进一步鉴定了AVN的解剖部位。6．α-actinin2对HEK293细胞Ik，ca的调节：共表达α-actinin2和SK2细胞的IK，ca电流密度明显大于仅表达SK2细胞的电流密度，Ik，ca电流密度增加了4倍。未转染的细胞，无明显IK，ca电流。Cytochalasin D预处理共表达α-actinin2和SK2通道的细胞，以阻断细胞骨架蛋白与SK2通道的相互作用，Ik，ca电流密度明显减小。7．SK2通道的表达及其与α--actinin2的共定位：Western blot检测共表达α--actinin2和SK2的HEK293细胞，其α--actinin2分子量约98 kDa。SK2蛋白表达为单聚体和二聚体，两个带分别约为60 and120 kDa。结果与期望值一致。激光共聚焦显微镜显示共表达SK2通道α-actinin2蛋白的HEK293细胞对SK2和α-actinin2抗体均具有免疫阳性反应，且SK2通道与α--actinin2共定位于HEK293细胞的细胞膜。结果提示SK2通道与α-actinin2分子的相互作用。讨论有关房室结电生理方面的动物研究见于家兔，偶见一例有关小鼠房室结电生理研究的报道。其原因在于房室结解剖部位的特点，房室结位于心房和室间隔之间，部位较深。此外，房室结细胞细小，且房室结的致密结区与周围结区之间包绕着结缔组织。由于这些特征，为电生理记录、免疫荧光染色、Western Blot等检测方法的应用带来了困难和限制，尤其是小动物。本研究成功建立了小鼠房室结制备和单个房室结细胞分离和鉴定方法，为从事小鼠靶基因模型，尤其是起搏功能异常的小动物研究奠定了基础。本研究采用两种靶基因模型，即过表达SK2和SK2通道敲除小鼠，使我们能够从整体水平、分离的房室结及单个细胞对SK2通道功能进行检测和比较。过表达SK2通道导致AVN细胞动作电位复极化加快，伴有AVN自发性电活动增加；而敲除SK2通道导致相反的AVN电活动变化。详细分析其变化，发现过表达SK2通道引起AVN着火率增加伴有增快的DDR和明显缩短的APD80。SK2通道敲除则引起AVN自发性电活动明显减慢，伴有明显延长的APD80和慢的DDR。为进一步证明SK2+／T和SK2+／△小鼠AVN功能的变化确实是由于SK2通道表达异常所致，本研究采用与记录心肌工作细胞相同的全细胞膜片钳程序，直接检测了AVN细胞IK，ca的变化。钳制电压-55mv使绝大部分存在于AVN细胞的一过性外向K⁺电流和T-型Ca²⁺通道失活，细胞外液无Na⁺和Ca²⁺，并用已知的细胞内Ca²⁺浓度激活SK2通道。在此条件下，可记录到与心肌工作细胞相近似的内向整流K⁺电流，并可被apamin所阻断。结果也表明过表达SK2通道，房室结细胞apamin-敏感K⁺电流明显增大，而敲除SK2通道，apamin-敏感K⁺电流几乎消失。SK2+／△小鼠AVN APD80明显延长可能与AVN细胞IK，ca下调有关，而SK2+／T小鼠AVN APD80缩短则可能与AVN细胞IK，ca上调有关。为研究SK2+／T小鼠和SK2+／△小鼠AVN功能异常是否与其SK2通道的表达相一致，采用免疫组织化学和激光共聚焦显微镜检测了房室结细胞SK2蛋白的表达。免疫组织化学证实房室结具有SK2免疫阳性反应。激光共聚焦显微成像表明房室结细胞对SK2抗体具有特异性的免疫反应，并表达于细胞膜。心房和心室细胞SK2亚单位表达于T管膜，因为心肌工作细胞存在T管系统（由细胞膜向细胞内凹陷而形成）与心肌兴奋-收缩耦联有关。微肌动蛋白（actinin）是肌动蛋白细胞骨架的结合蛋白。研究表明它可以连接细胞骨架蛋白与膜蛋白，例如膜离子通道。膜离子通道是高度特化的膜蛋白，其在细胞膜上的亚细胞定位是决定该通道功能的关键。肌型微肌动蛋白异构体α-actinin2可与一些膜离子通道结合并调节通道的功能，例如，调节电压-门控K⁺通道Kv1.5、NMDA受体、L-型Ca²⁺通道（Cav1.2）、Na⁺通道等。为进一步探讨SK2通道的作用机理，Chiamvimonvat实验室研究了SK2通道α-亚单位与其它蛋白的相互作用。酵母双杂交从人心脏的cDNA文库筛选并证明SK2通道C-末端有两个与α-actinin2的结合位点。体外生物化学、激光共聚焦显微镜也证明SK2通道与α-actinin2蛋白的相互作用及部分共定位。本研究表明SK2亚单位与α-actinin2共定位于HEK293细胞膜。α-actinin2可明显增加共表达SK2和α-actinin2细胞的Ca²⁺-激活K⁺电流。以往的研究表明L-型Ca²⁺通道与细胞骨架蛋白α-actinin2共定位，敲除Cav1.3通道可导致小鼠心肌细胞SK2通道下调。提示小鼠心房细胞SK2通道的功能取决于Cav1.3通道的正常表达，Cav1.3通道与SK2通道的共定位可能与α-actinin2-细胞骨架蛋白的相互作用有关。α-actinin2调节SK2通道可能通过不同的分子机理，一种可能机理即与钙调蛋白（Calmodulin，CaM）相关。α-actinin2和SK2通道C-末端都含有CaM结合区，且CaM是所有Ca²⁺-激活K⁺通道的感受器。已有研究表明CaM可与α-actinin2竞争与离子通道-NMDA受体结合，从而影响该通道的失活和在膜上的表达。已有报道细胞松弛素Cytochalasin D可直接影响微肌动蛋白与肌动蛋白细胞骨架的相互作用，加速心脏ATP-敏感的K⁺通道下调。本研究发现CyD可干扰共表达SK2和α-actinin2细胞的SK2通道功能。推测细胞骨架网络可能发挥某种作用将SK2通道与肌动蛋白细胞骨架连接并锚定在细胞膜。此外，还可直接或间接的调节通道的功能。本研究提出了一种新的途径研究并理解调节SK2通道的分子机理。SK通道的功能已被证明在于控制神经元的着火率。动作电位的触发，将引起细胞内Ca²⁺增加，进而激活SK能道，产生一持续时间较长的后超极化，后超极化的重要意义在于限制神经元的着火率和重复着火频率，保护细胞避免持续性和强直性兴奋。与SK通道在神经元的作用相比较，SK通道对心肌细胞的影响在于构成动作电位复极化的末期，此期相当于心脏兴奋性周期的相对不应期和超常期，临床上早搏或室上性心律失常常见这一时期。本研究证明心脏过表达SK2通道增加起搏细胞的着火率。推测当运动或交感神经反应性增高等生理情况下，SK2通道可增加房-室传导；而在病理情况下，过表达SK2通道，细胞内Ca²⁺增加也将明显影响房-室传导，例如，过表达SK2通道引起心房纤颤，其心房细胞上调IK，ca，引起快速复极化，加快房-室传导。因而，SK2通道在房性心律失常情况下对房-室传导有重要的调节作用。综上所述，本文首次证明了SK2通道过表达和SK2通道敲除小鼠存在明显的房室结功能异常。心脏SK2通道过表达可增加AVN细胞的自动着火率。而敲除心脏SK2通道则引起心动过缓，房-室传导减慢。心脏SK2通道过表达由于IK，ca上调，增加AP舒张期自动去极化和复极化速度。相反，敲除心脏SK2通道使IK，ca下调，进而减慢AP舒张期自动去极化和复极化速度。此外，本研究采用激光共聚焦显微镜，细胞生物学及电生理技术首次证明SK2通道与α-actinin2的分子相关。α-actinin2与SK2亚单位共定位于细胞膜，并调节SK2通道的功能。更多还原

【Abstract】 Background and objectiveThe atrioventricular node（AVN） is a highly specialized pacemaking tissue located at the junction of the right atrium and ventricle. AVN is the only electrical connection between atrium and ventricles and therefore it is responsible for transmitting electrical excitation from the sinoatrial node and atrium to working ventricular muscle. Electrical activity is conducted slowly through the AV node, which results in a delay between atrial and ventricular contraction, ensuriong that ventricular contraction does not begin before atrial contraction has ended. In some disease states, the AV node provides further functions. If the sinoatrial node or atrial conduction fails, the AV node becomes the ventricular pacemaker by its own spontaneous rhythm.Despite this vital roles, the electrophysiology of the AV node is still poorly understood. Studies have identified the roles of several ion channels in the AVN function and recent work has begun to identified an array of ion channel genes in the pacemaking tissues. Small-conductance Ca²⁺-activated K⁺ channels （SK channels） are voltage independent and are activated by intracellular Ca²⁺ ions. There are three SK channel subtypes （SK1-SK3） expressed in mammalian brain. SK channels are encoded by at least three genes: SK1, SK2, and SK3 （KCNN1, KCNN2, and KCNN3）, with differential sensitivity toward apamin, a selective SK channel antagonist. SK2 is highly sensitive to apamin, whereas SK1 channels are not affected by apamin. SK3 channels are intermediate.SK channels are widely expressed in different tissues, including the brain, peripheral nervous system, liver and smooth muscle. However, their presence and functional significance have not been well studied in the heart. Dr.Chiamvimonvan’s lab has recently identified SK channels in human and mouse cardiac myocytes are highly expressed in atrial compared to ventricular tissues. SK channels are important in the repolarization of cardiac action potentials in mice and humans. Furthermore, using genetically mouse model with over-expression of SK2 channel showed evidences of AVN and atrioventricular（AV） conduction dysfunction.In this paper, we test the functional properties of the AVN utilizing overexpression and knock-out of SK2 channel mice and develop an important information with regards to the functional roles of SK2 channel in AVN cells. Utilizing. HEK293 cells expressed SK2 channel, we investigated the molecular mechanisms of SK2 channel which is associated with a-actinin2, an important binding protein of actin cytoskeleton。Methods and Results1. Over-expressing of SK2 channel（SK2+/T）, knock-out of SK2 channel（SK2+/⊿）mice with C57B1/6J background and WT mice（16-22 weeks, both sexes） were used in this study.2. SK2+/T and SK2+/⊿ mice show evidence of SA and AV node dysfunctionSurface ECG recordings showed significant sinus bradycardia with prolongation of the PR intervals in SK2+/△mice compared to WT amimals.In contrast, SK2+/T mice showed significant shortening of the RR and PR intervals.These differences in the P-R and R-R intervals may represent abnormalities in the pacemaking activities in SA and AV nodal cells in the gene-targeted mice.3. SK2+/T nice show an increase the firing frequency of A VN while the opposite findings were observed in SK2+/⊿ miceThe mice were anesthetized by intraperitoneal injection of pentobarbital sodium （60mg/kg）. The heart was excised and the right atrium was opened. The AV node region was removed. Spontaneous action potentials（APs） were recorded from isolated AV nodal preparations from SK2+/T, SK2+/△, WT mice using microelectrode techniques with 3 mol/L KCl microelectrodes at 33℃.APs recorded from the AVN preparation can be identified by the presence of the slow diastolic depolarization and a very slow upstroke of phase 0. SK2+T mice showed a significant increase in the spontaneous activities of the AVN compared to WT controls.In contrast, SK2+/△mice showed a significant decrease in the firing frequency of the AVN compared to the WT group. Detailed analysis of the APs reveals significant changes in the CL, APD as well as DDR in the SK2+/T and SK2+/△mice. Over-expression of SK2 channel in SK2+/T mice resulted in a significant shortening of APD80, while APD80 was significantly prolonged in SK2+/△mice compared to WT animals.Moreover, SK2+/T mice showed a significant increase in the DDR and a corresponding shortening in the CL compared to WY control. The opposite effects were observed in SK2+/△mice.4. Single AVN cell Ik, ca recordingSingle AVN cells were isolated from WT, SK2+/T and SK2+/△mice as previously described with some modification. Whole-cell Ca²⁺-activated K⁺ current （Ik, ca）was recorded from single AVN cells at room temperature using patch-clamp technique. The current density elicited from a holding potential of-55mV, by the voltage-steps from-90mV to +40mV. Whole -cell current records Were filtered at 2 kHz and sampled at 10kHz. Apamin-semsitive K⁺ current were obtained using digital subtraction（before and after application of 500 pmol/L apamin）.The current density-voltage relationships of apamin-sensitive K⁺ current showed the inwardly rectifying currents which is decreased with increasing depolarization voltages. Both the inward and outward components of the Ik,ca were blocked by apamin. The reversal potential was -80mV, which is corresponding with Nernst equation.Over-expressing of SK2 channel in SK2+/T mice resulted in a significant increase in the apamin-sensitive current density in AVN cells and a significant decrease in the current density in the single AVN cells isolated from SK2+/△mice compared to WT animals. The results display evidence of the present of Ik, ca in the AVN cells.5. Double immunofluorescence labeling and Con focal MicroscopySingle isolated AVN cells from WT and SK2△/△mice were fixed with 4% paraformadehyde, then blocked with 1% BSA, incubated overnight at 4℃with primary antibodies. The following primary antibodies were used:（1）anti-SK2 antibody （1:100 dilution）& anti-neurofilamin （NF160 KD）antibody （1:100）;（2）anti-SK2 antibody （1:100）& anti-a-actinin2 antibody（1:800）.Confocal microscopy was performed by treatment with FITC-conjugated goat anti-rabbit antibody（1:250）and TEXRED-conjugated goat anti-mouse antibody（1:250）. Control experiments were performed by secondary antibody only under the same experimental conditions.Confocal microscopy illustrated the positive SK2 staining in single AVN cell as well as atrial and ventricular myocytes. NF160, a marker of the pacemaker and conduction system, was only expressed in AVN cell. The absence of positive SK2 staining was detected in the AVN cells in the SK2△/△mice.6. Expression of SK2 protein in WT mice AVNThe sections of the hearts from the WT and SK2△/△mice were blocked in 5% goat serum,then treated with anti-SK2 antibody（1:200）. The sections were then treated with biotinylated secondary antibody.Expression of SK2 protein in the AVN and working myocardium in WT animals was documented as brown positive staining and the absence of SK2 protein expression in the AVN in the SK2△/△mice was detected. AVN region was further evaluated in cardiac sections stained with H&E and Trichrome.7. HEK293 cell culture and plasmids transfectionHEK293 cells were used as a transient expression system. The cells were maintained in Dulbecco’s modified Eagle’s medium at 37℃in an air/5%CO₂ incubator. HEK293 cells were transfected using the following plasmids: 1) pSK2-IRES-EGFP & pcDNA3-a-actinin 2;2)pSK2-IRES-EGFP using Lipofectamine TM 2000 according to the manufacturer’s protocol. Whole-cell Ik, ca recorded from HEK 293 cells was performed at room temperature 36-48 hrs after transfection using the same recording protocol as for the AVN cells. The cells coexpressing SK2 channel plusα-actinin2 were pretreated with 2.5 pmol/L Cytochalasin D for 4 hrs before the recording.8. Modulation of Ik, ca by a-actinin 2 in HEK 293 cellsIk, ca density from HEK 293 cells expressedα-actinin 2 and SK2 channel was significantly larger than the current density from HEK 293 cells exresssed SK2 channel alone.Ik, ca density increase-4 fold when SK2 channel was co-expressed with a-actinin2 compared to SK2 channel expressed alone. No apamin-sensitive current was measured in non-transfected HEK 293 cells. Pre-treatment with cytochalasin D significantly reduced Ik, ca current density in HEK 293 cells expressed bothα-actinin2 and SK2 channel.9. Expession and subcellular co-localization of SK2 channel and a-actinin2 in HEK293 cellsFor transfected cells, immunostaining was performed 2-3 days post-transfection. The cells were fixed with 2% paraformadehyde, then blocked with 1% BSA, incubated overnight at 4℃with primary antibodies. The following primary antibodies were used:（1）anti-SK2 antibody （1:100 dilution）+anti-a-actinin2 antibody （1:800）.Confoeal microscopy was performed by treatment with FITC-conjugated goat anti-rabbit antibody（1:250）and TEXRED-conjugated goat anti-mouse antibody （1:250）. Control experiments were performed by secondary antibody only.Western blot documented the presence of a 98 kDa protein as expected forα-actinin2 in HEK 293 cells transfected withα-actinin2 and SK2 plasmids. The SK2 antibody recognizes a 60-kDa SK2 monomer and a higher molecular weight form, 120 kDa dimer.Confocal microscopy showed that SK2 channel andα-actinin 2 were co-localized in the plasma membrane of HEK 293 cells co-transfected with both SK2 andα-actinin 2 plasmids, suggesting thatα-actinin2 interacted with SK2 channel. Conclusion and DiscussionAlthough a few studies have described the electrophysiology of rabbit AVN, very little is known about the electrophysiological property of small animals AVN, including mouse AVN. The AVN is located at the junction of the right atrum and ventricular septum. There are fiber tissues around the compact node, which makes single cell isolation more difficult. Moreover, the small size of the AVN in mouse limites the study of single AVN cells using the electrophysiology, immunofluorescence, Western blot and so on. In this study, we describe an isolation procedure of mouse single AVN cells. Thus it offers considerable potential for the study of the gene-targeted mouse models including those which display abnormal pacemaker function.The two gene-targeted mouse models used in our study with over-expression and knock-our of SK2 channel allow us to directly test the functional roles of SK2 channel in the whole animal, AVN preparations as well as single isolated AVN cells. Overexpression of SK2 channels resulted in the increase in the repolarization of the AVN cells associated with an increase in the spontaneous activity. On the other hand, SK2 channel knock-out resulted in the opposite effects on the AVN activity. Detailed analysis of the spontaneous AP revealed significant changes in the APD and DDR in the SK2+/T and SK2+/△mice compared to WT control. The increasing firing frequency of the SK2+/T mice AVN is related to a significant shortening of APD80 and significant increasing in the DDR caused by over-expression of SK2 channel. The opposite effects were observed in the SK2 knock-out mice. The slow spontaneous activity of SK2+/△mice AVN is associated with a significant prolonged APD 80 and decrease DDR.To further document that the changes in the AVN observed in the SK2+/T and SK2+/△mice were indeed due to differing the expression of SK2 channel, we directly detected the Ik, ca measured as apamin-sensitive K⁺ current density in single isolated AVN cells from SK2 transgenic and knock-out animals using the same recording protocal as for the atrium myocytes. Whole-cell Ik, ca was recorded using voltage steps from a holding potential of-55 mV to inactivate the transient outward K⁺ currents, which were known to be present in mouse AVN myocytes. Na⁺ and Ca²⁺ ions were eliminated from the external solution. The interior of the cells was dialyzed using pipette solutions containing known concentrations of Ca²⁺. Using the above recording condition, a time-independent inward rectifier K⁺ current could be recorded. In addition, the component of both inward and outward current could be blocked by application of apamin. The results showed a significant increase in the apaminsensitive current density in AVN cells isolated from SK2+/T and a significant decrease in the current density in the SK2+/△mice compared to WT controls. The prolongation of ADP80 in the AVN in SK2 knock-out mice is due to downregulation of Ik, ca in the AVN, wherase the shortening of APD80 in SK2+/T mice upregulation of Ik, ca in the AVN.To determine whether the dysfunction of AVN in over-expression and knock-out SK2 channel is consistent with the expression of the SK2 channel, we detected the expression of SK2 subunite in heart AVN section and single AVN cells using immunohistochemistry and confocal. The expression of SK2 in AVN cells was localized to the surface membrane, whereas the immunofluorescence signal of SK2 channel in the atria and ventricular myocetes was localized to T-tubules which specialized regions of surface membrane which extend into the cell interior associated with the excitation-contraction of cardiac working myocytes.Actinin is members of a family of actin-binding cytoskeletal protein that may link the cytoskeleton to membrane proteins such as ion channel. Previous work has demonstrated that ion channels undergo highly specialized subcellular localization at the plasma membrane which is crucial to cell function. Muscle actinin isoformα-actinin2 binds and modulates the functions of a voltage-gated K⁺ channel, Kv1.5, NMDA-type glutamate receptor, L-type Ca²⁺ channel（Cav1.2）and so on.To seek molecular that bind toα-subunit of the SK2 channel, yeast two-hybrid system revealed the interaction of SK2 channel c-terminus withα-actinin2. In vitro biochemical methods and confocal were applied to confirm the interactions betweenα-actinin2 protein and SK2 channel. Our data showed the functional significance of the interaction betweenα-actinin2 and SK2 channel. The Ik, ca density from HEK 293 cells transfected with bothα-actinin2 and SK2 channel was significantly large than current density from HEK 293 cells transfected with SK2 channel alone. Furthermore, L-type Ca²⁺ channels were found to be co-localized with a-actinin2 cytoskeletal protein. Knock-out Cav1.3 channel in the heart resulted in downregulation of SK2 channel in the mouse cardiac myocytes. The functions of SK2 channels in the heart are dependent on normal expression of Cav1.3 channel in mouse atrial myocytes. The co-localization of SK2 channel with Cav1.3 channel may be associated with theα-actinin2 cytoskeletal protein.α-actinin2 modulates the SK2 channel by different molecular mechanisms. One possible molecular is Calmodulin（CaM）. Bothα-actinin2 and SK2 C-terminus contain a CaM binding domain. Moreover, CaM is a Ca²⁺ sensor for all of Ca²⁺-activated K⁺ channels. Indeed, one study has demonstrated that CaM is known to compete withα-actinin2 for binding to another ion channel, the NMDA receptor, thereby mediating both inactivation of the channel and their localization.Evidence indicated that Cytochalasin D accelerated cardiac ATP-sensitive K⁺ channel rundown and directly influence the interaction between actin and actinin. Indeed, we found that Cytochalasin D modulated SK2 channel function in the transfected HEK293 cells. Cytoskeletal networks may exert an interaction with SK2 protein, directly and indirectly modulate its channel function, in addition to help its anchorage to the membrane. This study points the way to new regulatory mechanisms for cardiac ion channel.SK channels have been shown to play an important role in setting the tonic firing frequency of neurons. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the frequency of repetitive APs. The increase in intracellular Ca²⁺ evoked by AP firing allows SK channels activation to generate a long lasting hyperpolarization. This hyperpolarization protects the cell from the deleterious effects of continuous tetanic activity. In contrast to the hyperpolarization effects of SK channels in neuron, in cardiac myocytes, the SK channels contributed markedly toward the late phase of the cardiac repolarization which is susceptible to extra excitation, e.g. early after depolarization and arrhythmias. Here, we demonstrate that over-expression of SK2 channel in the heart increases the firing frequency of the pacemaking tissues. Therefore, the SK2 channel may serve a distinct role to potentiate the increase in AVN conduction during exercise or sympathetic responses under normal physiological condition. On the other hand, an increasing intracelluar Ca²⁺ under pathologic conditions may produce profound changes in AVN conduction. For example, during atrial fibrillation, the rapid depolarization may increase intracellular Ca²⁺ and potentiate the Ik, ca and AVN conduction. Hence, SK2 channel may modulate atrioventricular conduction during atrial arrhythmias.Take together, here we first documented the AVN dysfunction in the SK2+/T and SK2+/△mice. Over-expression of SK2 channel in the heart increased the frenquency of firing of the AVN. Knock-out of SK2 channel in the heart caused bradycardia and delays atrioventricular （AV）conduction. Furthermore, over-expression of SK2 channel in the heart increased the rates of diastolic depolarization and repolarization due to upregulation of Ikca in the AVN. Knock-out of SK2 channel in the heart slowed the rates of diastolic depolarization and prolonged the duration of repolarization because of downregulation of Ikca.In addition, in the present study, we first employed confocal immunohistochemical and eletrophysiological techniques to demonstrate that the association of a-actinin2 with SK2 channel localizes the channel to the membrane and regulates the channel function. Our data present the first study on the molecular mechanism of coupling of SK2 channel with cytoskeletal proteins. 论文部分小电导-Ca（2+）-激活K⁺通道在小鼠房室结细胞的表达及其功能的研究#1引言#1第一部分SK2通道在小鼠房室结细胞的功能性作用#31．前言#32．材料和方法#33．实验结果#84．讨论#17参考文献#22第二部分α-actinin2对SK2通道的功能性调节#271．前言#272．实验材料和方法#283．实验结果#324．讨论#36参考文献#38综述部分小电导-Ca²⁺-激活K⁺通道#41参考文献#54后记缩略词表#66读博期间发表的论文#68致谢#69更多还原