Activation of a Na+/H+ exchange at the plasma membrane of sea urchin gametes is responsible for triggering motility in sperm and initiating development in fertilized eggs. The goal of this work is to understand the mechanism of the exchange process and the molecular events leading to its activation. Studies from this laboratory indicate the Na+/H+ exchange system in sperm flagellar membranes is sensitive to membrane potential and is turned off by depolarization. This unique voltage-gating mechanism will be characterized further. The kinetics of the exchange will be resolved. The 22Na+/Na+ exchange will be measured. The symmetry of the gating mechanism with respect to the polarity of the membrane potential will be determined. The Na+-binding site of the exchanger will be probed and labeled by group-specific chemical modification techniques. Finally, the flagellar exchanger will be solubilized and reconstituted. Mechanisms involved in the activation of the Na+/H+ exchange in eggs will be examined using plasma membrane preparations derived from eggs and zygotes. Since protein kinase C appears to be involved, the enzyme will be purified and characterized. The intracellular distribution of kinase C will be determined by subcellular fractionation. The endogenous membrane proteins phosphorylated by kinase C will be identified. The effects of phosphorylation on the Na+/H+ exchange activity will be studied in isolated membrane vesicles. The role of polyphosphoinositides (PPI) breakdown in the activation process will be investigated in vivo. Neomycin, an inhibitor of PPI breakdown will be injected into eggs, and these eggs will be monitored for effects on Ca++ mobilization and Na+/H+ exchange produced by fertilization or activation by A23187 and phorbol esters. The possible role of GTP-binding protein in the PPI breakdown will be examined. Finally, coated-vesicle endocytosis will be investigated as a possible means for retrieving the Na+/H+ exchange from the plasma membrane of the zygotes.
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