Calcium levels in cells are controlled in part by the plasma membrane calcium ATPases. The calcium pump is highly conserved from paramecia to humans and has many sites of control including phosphorlation by protein kinases and binding calmodulin. Mutations in this plasma membrane pump have been shown to cause such diseases as deafness and balance deficits. We are interested in its role in chemoresponse. We have found indirect evidence that activation of the calcium pump figures into the transduction pathways for chemoreception in Paramecium. (See evidence described in the proposal.) I propose to examine the regulation of the Paramecium calcium pumps and their potential roles in chemosensory signal transduction. Specifically, I plan to test the hypotheses that the calcium pump is activated by phosphorylation of and by calmodulin binding to the autoinhibitory domain (also called calmodulin binding domain, or CBD) in two different chemosensory pathways. In preparation, I have created CBDs in which two conserved serines are replaced by alanines (AA) or glutamates (EE). As expected, the wild type CBD is phosphorylated by protein kinases A and C in vitro, but the mutant AA and EE CBDs were not. Calmodulin binds to the wild type and AA CBD, but not to the EE mutant. I will use the CBDs as specific inhibitory reagents to probe for the role of phosphorylation and calmodulin binding in chemosensory signal transduction and to test my hypotheses.
