E/C COUPLING AND CALCIUM REGULATION IN ATRIAL MYOCYTES

提出的研究的总体目标是表征[Ca2+]i调节和肌浆网（SR） Ca2+释放机制在兴奋-收缩（e-c）偶联在哺乳动物心房肌。我们将检验心房细胞正常e-c偶联涉及钙离子从连接（j-SR）和非连接SR （nj-SR）释放的假设。然而，只有j-SR的释放直接依赖于膜电压（即通过Ca2+通过电压门控Ca2+通道进入触发Ca2+诱导的Ca2+释放（CICR）），而nj-SR的释放仅由Ca2+和CICR的扩散触发（通过类似于心脏[Ca2+]~波传播的机制）。在心房肌中，e-c耦合的“局部控制”模型严格适用于j-SR的释放。SR Ca2+的释放受到磷酸化-去磷酸化循环的严格调控，ATP在SR释放通道的微结构域中以糖酵解的方式产生。代谢诱导的机电和[Ca2+]1瞬态交替是心房心律失常的主要危险因素，将为研究区隔化糖酵解ATP形成对e- c偶联的动态调节提供模型系统。拟建研究的五个主要具体目标是：1。表征由动作电位触发的哺乳动物心房细胞全细胞[Ca2+]1瞬态的时空特性。2. 验证心房细胞e-c偶联局部控制模型的有效性，表征j-SR和nj-SR e-c偶联和Ca2+释放的机制。3. 定量定义心房细胞中j-SR和nj-SR Ca2+释放（Ca2+火花）的基本事件的性质。4. 表征代谢诱导的[Ca2+]i交替及其细胞机制。5. 确定区室化糖酵解ATP产生在调节SR Ca2+释放和CICR中的作用。为了实现这些目标，将使用多种实验技术，包括在单个心房肌细胞中使用激光扫描共聚焦显微镜进行高分辨率[Ca2+]成像，研究膜电流的全细胞电压钳技术，通过心脏SR Ca2+释放通道重建成平面脂质双层的单通道记录，通过2光子激发对笼中Ca2+的亚细胞光解，以及Ca2+进入，释放和摄取的药理学操作。提出的研究将提供基本的新信息心房e-c偶联和Ca2+释放在正常和改变条件下与心房心律失常相关。

The overall goal of the proposed study is to characterize [Ca2+]i regulation and sarcoplasmic reticulum (SR) Ca2+ release mechanisms during excitation-contraction (e-c) coupling in mammalian atrial muscle. We will test the hypothesis that in atrial cells normal e-c coupling involves Ca2+ release from both junctional (j-SR) and non-junctional SR (nj-SR). However only release from j-SR is directly dependent on membrane voltage (i.e. via Ca2+ entering through voltage-gated Ca2+ channels triggering Ca 2+-induced Ca2+-release (CICR)), whereas release from nj-SR is triggered solely by diffusion of Ca2+ and CICR (by a mechanism similar to cardiac [Ca 2+]~ wave propagation). In atrial muscle the model of 'local control' of e-c coupling strictly applies only to release from j-SR. SR Ca2+ release is tightly regulated by a phosphorylation-dephosphorylation cycle for which ATP is produced glycolytically in the microdomain of the SR release channel. Metabolically-induced electromechanical and [Ca2+]1 transient alternans, a major risk factor for atrial arrhythmias, will provide a model system to study the dynamic regulation of e- c coupling by compartmentalized glycolytic ATP formation. The five major specific aims of the proposed research are: 1. Characterize the spatio-temporal properties of whole-cell [Ca2+]1-transients in mammalian atrial cells triggered by action potentials. 2. Test the validity of the local control model for e-c coupling in atrial cells and characterize the mechanisms of e-c coupling and Ca2+ release from j-SR and nj-SR. 3. Define quantitatively the properties of elementary events of Ca2+ release (Ca2+ sparks) from j-SR and nj-SR in atrial cells. 4. Characterize metabolically-induced [Ca2+]i alternans and its cellular mechanisms. 5. Define the role of compartmentalized glycolytic ATP production in modulating SR Ca2+ release and CICR. To achieve these aims a multitude of experimental techniques will be used, including high resolution [Ca2+]i imaging by laser scanning confocal microscopy in single atrial myocytes, whole- cell voltage clamp techniques to study membrane currents, single channel recordings through cardiac SR Ca2+ release channels reconstituted into planar lipid bilayers, subcellular photolysis of caged Ca2+ by 2-photon excitation, and pharmacological manipulation of Ca2+ entry, release and uptake. The proposed research will provide fundamental new information on atrial e-c coupling and Ca2+ release under normal and altered conditions relevant to atrial arrhythmias.