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

拟议的研究将解决膜运输过程的特征，位于Chara和Nitella的质膜上，沿着这些巨型藻细胞的长度产生细胞外电流模式。根据这些细胞外电流在光激活条件下获得的初步电流-电压分布，以及在pH脉冲实验中获得的这些电流的振动探针测量，提出了酸带和碱性带是由II类输运系统产生的假设。现在将对这一假设进行广泛的生物物理测试，使用最先进的电流电压测量和动力学建模相结合。在这些研究中，一种新的计算机操作的二维振动探针系统将提供关于对假定的II类转运体进行时空控制的细胞机制的重要信息。离子选择微电极也将用于记录光诱导的细胞质钙和pH的变化，旨在阐明照明后这些膜运输过程的时间依赖性激活的生化过程。该假设将被验证，暗诱导的运输失活是通过一个耦合反应来实现的，包括细胞质钙的增加，激活钙-钙调素，然后钙-钙调素激活蛋白质磷酸化酶，通过磷酸化使氢离子运输系统失活。蛋白质磷酸酶在再激活中的作用也将被研究。最后，通过抑制叶绿体和线粒体的70S和细胞质的80S蛋白质合成，进一步研究蛋白质合成在调控过程中的作用。巨藻Chara和Nitella多年来一直是生理学研究的主题。它们细胞的大尺寸和它们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绝佳系统。这项研究的结果使用了最先进的生物物理技术，应该大大提高我们对植物细胞膜生理学的理解。

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.