| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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
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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
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