DESCRIPTION: (Applicant's Abstract) It is currently believed that much of the regulation and modulation of the Na,K,Cl cotransporter (NKCC) involves phosphorylation of the NKCC protein. Using the internally dialyzed squid giant axon, we have recently made two novel phosphorylation-related observations about the NKCC. In the nominal absence of ATP and ADP, arginine phosphate (Arg-P) supports NKCC fluxes across the squid axolemma. We will pursue this unexpected finding by testing whether Arg-P will support phosphorylation of the NKCC using a squid-specific antibody (which we will develop) to perform immunoprecipitation studies. We will test for whether the effect of arginine phosphate may be due to the replenishment of a compartment of ATP which is poorly accessible to dialysis, but readily accessible to the NKCC. We will perform a series of functional studies to determine whether the Arg-P activated NKCC behaves qualitatively different from the ATP activated NKCC. A second unexpected finding involves the ability of two inhibitors (A3 and DRB) of protein kinase CK2 (aka casein kinase 2) to completely block NKCC-mediated ion fluxes. This is particularly interesting given that the squid NKCC and those from mammalian sources have numerous CK2 consensus phosphorylation sites. To date, we have not identified any other types of protein kinase inhibitors capable of inhibiting the squid NKCC. We will take advantage of the fact that CK2 uses GTP almost as effectively as ATP to test whether GTP can support NKCC fluxes. We will again use the squid-specific antibody to test for whether treatment with A3 or DRB reduces the phosphorylation of the NKCC. Axonal shrinkage stimulates the squid NKCC by shifting the intracellular Cl- inhibition curve toward higher [Cl-]I values. We will test for roles for CK2 and for actin polymerization in NKCC activation by shrinkage. Axonal shrinkage leads to an increase of axoplasmic ionic strength which is known to facilitate the polymerization of actin. A series of studies examining the effects of several known modulators of F-actin on NKCC-mediated flux will be performed to test for a role for actin polymerization in activation of the NKCC. Another series of studies will be directed toward obtaining the functional expression of a clone of the squid NKCC using both mammalian cell and Xenopus oocyte expression systems. We will also develop a cut-open oocyte preparation for the characterization of the cloned squid NKCC. It will be used to functionally compare the squid NKCC with that from mammalian sources. In addition, the cut-open oocyte model will permit the year-round study of squid NKCC in a preparation that gives reasonably good access to intracellular regulatory sites on the NKCC protein.