Ca-dependent Cl channel in tracheal smooth muscle and airway hypersensitivity

在离体豚鼠气管平滑肌细胞上，我们比较了去极化或咖啡因激活的Ca^2+依赖性Cl^-电流与Ca^2+依赖性K^+电流的时间和Ca^2+依赖性。K^+电流的激活比Cl^-电流的激活快，如果将膜电位固定在一个稳定的水平上，K^+电流的激活仅依赖于质膜下Ca^2+浓度，而不依赖于时间。另一方面，Cl^-电流激活表现出时间依赖性。在该项目的后期，进行了Ca^2+动员和膜电流图像的同时测量。用激光共聚焦荧光显微镜（Nikon RCM 8000），通过膜片式移液管的简单扩散，从负载indo-1或fluo-3的单细胞中获得Ca^2+图像。结果发现，Ca^<2+>依赖性Cl^-电流的激活慢于全身Ca^<2+>浓度的升高。 ...更多信息 输出100毫秒。最近有研究表明，钙调素依赖性蛋白激酶II对Cl^-通道蛋白的磷酸化参与了气管平滑肌Ca^2+依赖性Cl^-通道的失活。基于激活的缓慢时间过程，很难解释Ca^<2+>与通道直接结合的激活。然而，药理学结果表明，通道磷酸化的主要贡献之一的蛋白激酶的激活是不太可能的。此外，我们还尝试在有皮肤的单细胞中，在细胞上的膜片配置下记录负责Ca^2+依赖性Cl^-的单通道电流，其中质膜被透化，但细胞内信号转导系统保持可用。在由内而外的贴片结构中，通道电流不能被很好地记录。在贴细胞膜片下，记录到大电导的Ca^2+依赖性Cl^-通道电流。由于单通道电流的特性与基于宏观Cl^-电流的预期有些不同，因此可以假设存在另一种类型的Cl^-通道，其电导可能更小。少

The time and Ca^<2+> dependence of Ca^<2+> dependent Cl^- currents, which were activated by depolarization or caffeine, was examined in comparison with that of Ca^<2+> dependent K^+ current in single smooth muscle cells isolated from guinea-pig trachea. The activation of the K^+ current was faster than that of the Cl^- current, simply dependent on subplasmalemma Ca^<2+> concentration if the membrane potential was clamped at a steady level, and not dependent on time. On the other hand, the Cl^- current activation showed time-dependence. In the later part of the project, the simultaneous measurement of images of Ca^<2+> mobilization and membrane currents were performed. Ca^<2+> images were obtained from single cells which wereloaded with indo-1 or fluo-3 by simple diffusion from patch pipettes using a laser confocal fluorescent microscope (Nikon RCM8000). It was found that the activation of Ca^<2+> dependent Cl^- current was slower than the elevation of global Ca^<2+> concentration by ab … More out 100 msec. It has been reported recently by other research group that phosphorylation of the Cl^- channel protein by calmodulin-dependent protein kinase II is included in the inactivation of the Ca^<2+> dependent Cl^- channel in tracheal smooth muscle. Based on the slow time course of activation, it is rather difficult to explain the activation by the direct binding of Ca^<2+> to the channel. Pharmacological results, however suggest that the major contribution of channel phosphorylation by one of the protein kinases to the activation is unlikely. Moreover, the recording of single channel current responsible for the Ca^<2+> dependent Cl^- was tried under on-cell patch configuration in skinned single cells, where plasma membrane was permeabilized but intracellular signal transduction systems were kept available. In inside-out patch configuration, the channel current can not be recorded well. Under the on-cell patches, a large conductance Ca^<2+> dependent Cl^- channel currents were recorded. Since characteristics of the single channel current was somewhat different from those expected based on the macroscopic Cl^- currents, the existence of another type of Cl^- channel which may have smaller conductance can be assumed. Less