Mechanism and Regulation of the Transport Processes Responsible for the Extracellular Current Pattern Formation in Chara and Nitella

这项拟议的研究将探讨位于轮藻和尼特拉细胞质膜中的膜运输过程的特征，这些过程产生沿这些巨大藻细胞长度的细胞外电流模式。根据这些胞外电流处于光激活状态时获得的初步电流-电压分布，以及在pH脉冲实验期间对这些电流的振动探针测量，提出了酸带和碱性带是由II类运输系统产生的假设。现在将使用最先进的电流-电压测量结合动力学模型对这一假设进行广泛的生物物理测试。对于这些研究，一种新的计算机操作的二维振动探头系统将提供关于负责对假定的II类转运体进行时间和空间控制的细胞机制的重要信息。离子选择性微电极也将被用来记录光诱导的细胞质钙和pH的变化，旨在阐明这些膜运输过程在光照后依赖于时间的激活的生化过程。这一假说将被验证，黑暗诱导的运输失活是通过一个耦合反应来实现的，该反应包括胞浆内钙的增加激活钙-钙调蛋白，然后钙-钙调蛋白激活一种蛋白磷酸化酶，通过磷酸化使氢离子运输系统失活。还将调查蛋白磷酸酶参与再激活的情况。最后，将使用70S(叶绿体和线粒体)和80S(细胞质)蛋白质合成的抑制剂进一步研究蛋白质合成在调节过程中的参与。多年来，大型藻类轮藻和尼特拉一直是生理学研究的对象。它们的细胞大小和产生的依赖于光的电流模式使它们成为研究细胞电现象背后的膜运输过程的极佳系统。这项使用最先进的生物物理技术的研究结果应该会极大地增强我们对植物细胞膜的生理学的理解。

The proposed research will address the characterization of the membrane transport processes, located in the plasma membrane of Chara and Nitella, that generate the extracellular current pattern along the length of these giant algal cells. Based on preliminary current-voltage profiles, obtained when these extracellular currents were in the light-activated condition, and on vibrating probe measurements of these currents obtained during pH pulse experiments, the hypothesis has been proposed that the acid and alkaline bands are generated by a Class II transport system. An extensive biophysical test of this hypothesis will now be conducted using state of the art current-voltage measurements in combination with kinetic modelling. For these studies a new computer-operated 2-dimensional vibrating probe system will provide important information on the cellular mechanisms responsible for temporal and spatial control over the putative Class II transporter. Ion selective microelectrodes will also be used to record light-induced changes in cytoplasmic calcium and pH in experiments aimed at elucidating the biochemical processes responsible for the time dependent activation of these membrane transport processes following illumination. The hypothesis will be tested that dark-induced inactivation of transport is effected through a coupled reaction involving an increase in cytoplasmic calcium which activates calcium-calmodulin followed by calcium-calmodulin activation of a protein phosphorylase which inactivates the hydrogen ion transport system by phosphorylation. The involvement of a protein phosphatase in reactivation will also be investigated. Finally, the involvement of protein synthesis in the regulation process will be further investigated using inhibitors of 70S (chloroplast and mitochondria) and 80S (cytoplasmic) protein synthesis. The giant algae Chara and Nitella have been the subject of physiological studies for many years. The large size of their cells and the light dependent patterns of electrical current that they generate make them an excellent system in which to study the membrane transport processes underlying cellular electrical phenomena. The results of this research using state of the art biophysical techniques should greatly enhance our understanding of the physiology of plant cell membranes.