Na+/H+ exchanger is a broadly distributed transporter that plays a central role in a variety of cellular processes including cell volume and intracellular pH regulation. The same trasporter has also been implicated in a variety of pathological conditions including hypertension, diabetes, and the response of several tissues to hypoxic/ischemic insults. The combination of several isoforms of the transporter and their sensitivity to a variety of controlling mechanisms, have been frequently invoke as an explanation for the versatility of the exchanger and its involvement in many (and sometimes contradictory) biologicalprocesses. In this application, we propose to use the Amphiuma erythrocyte as a model system to study the regulation of this inducible transporter. Evidence fromthis and other laboratoris indicated that the activity of the transporter is regulated by protein phosphorylation, suggesting a role for protein kinases and phosphatases inthis regulation. Our working hypothesis is that: """"""""Interactions between several protein kinases and phosphatases, acting directly on the exchange protein or indirectly on a closely associated protein, ae responsible for the regulation of the volume sensitive Amphiuma erythrocyte Na+/H+ exchanger"""""""". In order to test this hypothesis we are proposing to expand our pharmacological and biochemical approach to establish the role, and cross-talk interactions, between several signaling pathways in regulating exchange activity. Specifically, the proposed study will focused on the following kinases: casein kinase-II, protein kinase-C, tyrosine kinases, mitogen activated protein kinases, and Ca++- calmodulin dependent kinase. The role and interaction of protein phosphatases will also be established, and the ability of these kinases and phosphatases in modulating the net phosphorylation of the exchanger will be determined by immunoprecipitation studies. Emphasis will be given to the cross-talk interactions between the different signaling cascades and its net effect on exchange activity. Finally, the possibility of regulation of the exchanger by recuritment-removel of transporter to/from the plasma membrane by fluid-phase endocytosis will also be explored. Identification and characterization of the vesicles and the co-migration of the exchange protein in these organelles willbe established with the use of antibodies, in combination with confocal microscopy, and in isolated vesicles followed by immuno-blot analysis. The results of these studies shold provide fundamental information concerning the mechanism that regulate Na+/H+ exchange function, and will have broad implications for cellular physiology and pathophysiology.
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