Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle.
Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle.
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DOI：
10.1085/jgp.200609519
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发表时间：
2006-06
影响因子：
3.8
通讯作者：
Santana, Luis F
中科院分区：
文献类型：
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作者：
Navedo, Manuel F;Amberg, Gregory C;Nieves, Madeline;Molkentin, Jeffery D;Santana, Luis F
在动脉平滑肌中，单个或小簇的Ca 2+通道以高概率模式运行，产生几乎连续的Ca 2+内流位点（称为“持续性Ca 2+火花”位点）。持续的Ca 2+火花活性在任何给定的细胞内区域性地变化。目前，Ca 2+通道的分子身份的Ca 2+火花和机制，使其空间异质性仍然不清楚。在这里，我们使用全内反射荧光（TIRF）显微镜来直接调查这些问题。我们发现表达L型Cavα1.2通道的tsA-201细胞重现了脑动脉肌细胞中Ca 2+火花的一般特征，包括量子事件的幅度、电压依赖性、门控模式和药理学。此外，PKCα活性是动脉肌细胞和tsA-201细胞中基础持续性Ca 2+火花活性所必需的。在动脉肌细胞中，蛋白磷酸酶2A（PP 2A）和2B（PP 2B;钙调神经磷酸酶）的抑制通过引起新的持续性Ca 2+火花位点和通过增加先前活性位点的活性来增加Ca 2+内流。PP 2A和PP 2B抑制Ca 2+火花的作用需要PKC活性，表明这些磷酸酶反对PKC介导的磷酸化。总之，这些数据明确地表明，持续的Ca 2+火花活动是一个基本的L-型Ca 2+通道时，与PKC的属性。我们的研究结果支持了一种新的模型，其中L型Ca 2+通道的门控模式在细胞内区域性变化，取决于附近PKCα，PP 2A和PP 2B的相对活性。
In arterial smooth muscle, single or small clusters of Ca2+ channels operate in a high probability mode, creating sites of nearly continual Ca2+ influx (called “persistent Ca2+ sparklet” sites). Persistent Ca2+ sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca2+ channels underlying Ca2+ sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavα1.2 channels recapitulated the general features of Ca2+ sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCα activity was required for basal persistent Ca2+ sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca2+ influx by evoking new persistent Ca2+ sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca2+ sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca2+ sparklet activity is a fundamental property of L-type Ca2+ channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca2+ channels vary regionally within a cell depending on the relative activities of nearby PKCα, PP2A, and PP2B.