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

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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.