CALMODULIN KINASE II AND ION TRANSPORT IN THE KIDNEY

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Calcium-calmodulin dependent protein kinase II) (CaM-KII) is a widely distributed enzyme with a broad substrate specificity. Recent studies from our laboratory have demonstrated the presence of CaM-KII in cells of the rabbit renal proximal tubule. The presence of MRNA for alpha subunit of CaM-KII visualized by Northern hybridization analysis a protein of appropriate molecular size by Western immunoblot analysis using an antipeptide antibody against CaM-KII, and CaM-KII protein kinase activity itself were established. The effect of activation of this protein kinase on electrolyte transport in the renal proximal tubule is not well defined. The present proposal describes experiments designed to study the effect of CaM-KII on electrolyte transport in renal brush border and basolateral membranes and the regulation of the protein kinase in the renal proximal convoluted tubule. Particular emphasis will be placed on the effect of CaM-KII on transport systems which mediate hydrogen ion, bicarbonate, and sodium transport in the renal proximal tubule. The rationale for this is derived from recent studies indicating that phosphorylation by CaM-KII inhibits the solubilized and reconstituted Na + -H+ exchanger. Recent studies by others have also raised the possibility that CaM-KII regulates the activity of sodium dependent bicarbonate co-transport in addition to Na+-H+ exchange transport in the basolateral membrane of proximal tubule cells. The possible effects of this protein kinase on other proximal tubule functions have not been studied. Moreover, no studies to date have examined regulation of this protein kinase in renal proximal tubules. The present proposal employs some newly developed techniques which obviate some of the prior experimental difficulties involved in the study of the biologic effects and the regulation of CaM-KII. To study the effect of CaM-KII on renal electrolyte transport, renal brush border (BBM) and basolateral accomplished through transiently opening the vesicles by exposure to hypotonic solutions containing ATP, Mg2+,Ca2+, calmodulin, and CaM-KII. The activities of Na+-H+ exchanger, Na+-dependent glucose transporter, and the Na+PO'4 transporter in BBM in addition to Na+- HCO3 co-transporter and Na+-K+ ATPase in BLM will be measured using established methods. This experimental approach, which has been employed previously to study the effects of PKA and PKC, should provide new information on the regulatory role of CaM-KII in the control of ion transport in the renal proximal tubule. CaM-KII may be involved in regulation of proximal tubule function by hormones whose signal transduction pathways are linked to changes in intracellular calcium levels. Therefore, utilizing isolated proximal convoluted tubules, we will examine the effect of dopamine or angiotensin II treatment on the autophosphorylation and activation of 50 Kda subunit of renal CaM-KII, and the expression of CaM-KII MRNA by Northern hybridization and slot blot analyses using a CDNA to the alpha subunit of rat brain CaM-KII activity in extracts from the suspended proximal convoluted tubules will also be assayed using specific peptide substrates. These experiments should provide new information on the regulation of this protein kinase in the renal proximal convoluted tubule.