This is a proposal to study two different chloride transport mechanisms; both of which appear to be widely distributed in animal cells. One mechanism uses movements of C1, Na and HCO3 to extrude or neutralize intracellular acidity. In this proposal we will examine how this system is activated by acidic intracellular pH and how it changes from an apparent C1/C1 exchange mechanism to a more complicated Na and HCP3 - dependent acid extruder. Our central hypothesis is that ATP-dependent phosphorylation plays a key role in these events. We will perform detailed studies of ion transport to obtain quantitative kinetic information about this transport mechanism. The second chloride transport mechanism to be studied is the Na, K, Cl co-transporter. This transporter also requires ATP but presumably not for energetic or thermodynamic reasons. We believe phosphorylation is also involved for the regulation of activity by this transporter. In both transporters we will study fundamental transport properties as well as the effects of agents to stimulate and/or inhibit protein kinases and protein phosphatases in an effort to substantiate our hypothesis of transport activation via protein phosphorylation. These studies will be conducted on giant cells (squid giant axon and barnacle giant muscle fibers). Their large size allows us to use the technique of intracellular dialysis to control both intra-and extracellular contents. The method permits the measurement of unidirectional ion fluxes by these chloride transport mechanisms, we will use radioactive-isotope-labelled ligands to attempt to label and partially identify the membrane protein(s) involved in the transport processes.
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