Transient shifts in the intracellular calcium concentration can initiate a variety of developmental and physiological changes in many nonexcitable cell types. This project is centered on the involvement of calcium channels in lymphocyte and platelet activation. The calcium influx portion of the signal will be analyzed using a fluorescent probe of intracellualr calcium concentration. Experiments will test the hypothesis that calcium movement is facilitated by a ligand-activated single-file channel, with multiple calcium binding sites providing the specificity, and that control of the plateau height in the cell is separate from the number of channels that are activated initially to create the signal. Temperature dependence will show whether the influx is relatively independent of membrane fluidity, and competition between cations may provide evidence for multiple calcium binding sites in the ion channel. An extension of Indo-1 methodology will be used, employing whole spectra instead of only two fluorescence emission wavelengths. This approach will extend the applicability of the dye to multiple cations. Ionic movement across the plasma membrane has been attributed as being causal in the activation of lymphocytes and platelets. Changes in the cytosolic level of calcium have been determined to be essential to the activation of these cells. This project focuses on the role of the class of membrane proteins known as channels in the facilitated diffusion of calcium ions into these cells during activation. The experiments will employ calcium-indicator dyes, whose changes in fluorescence emission report on small and rapid changes in the free concentration of calcium after the channels open during cell activation. The major aim of this project is directed toward the characterization of the channels responsible for the sustained increase in cytosolic calcium activity that, in turn, underlies early stages of lymphocyte and platelet activation.
