engleski

Calcium/calmodulin dependent protein kinase II constitutively binds and regulates the Ileal BB Na+/H+ exchanger NHE3

Calcium/calmodulin dependent protein kinase II Constitutively Binds and regulates the Ileal BB Na+/H+ Exchanger NHE3 M. Zizak#, D. Bartonicek#, B. Cha$, R. Murtazina$, C.M. Tse and M. Donowitz$ From the # Department of physiology, School of Medicine, University of Zagreb, Croatia and the $ Department of gastroenterology, The Johns Hopkins University, Baltimore, Maryland We previous showed that Ca2+-calmodulin and calcium/calmodulin dependent protein kinase II (CaMKII) was involved in regulation of ileal neutral NaCl absorptive process and its component brush border Na+/H+ exchanger NHE3. We now investigate the relationship between CaMKII and NHE3 at a molecular level using PS120 cell fibroblasts expressing NHE3. We showed by using in vivo co-immunoprecipitation that CaMKII binds in intact cells. This binding occurs within a discrete part of the NHE3 C-terminal domain at a.a. 585-605 under basal condition. Furthermore, "pull down" assay using fusion proteins containing different parts of the NHE3 cytoplasmic domain ; F1 (a.a. 475-581), F2 (a.a. 581-667), F3 (a.a. 667-744), and F4 (a.a. 744-832) demonstrated that CaMKII bounds only to the F2 fusion protein. Moreover, "pull down" assays showed that CaMKII directly bounds to NHE3 and that this binding was calcium independent to NHE3. Na/H exchange activity was measured fluorometrically to determine role of CaM kinase in NHE3 regulation. KN-62, a CaMKII inhibitor, increased Na+/H+ exchange activity for ~33% demonstrating that CaMKII inhibits basal NHE3 activity. A negative control KN04 did not alter NHE3 activity. The KN-62 effect was evaluated in cells in which Ca2+ was elevated with the Ca-ATPase inhibitor thapsigargin. KN-62 stimulated NHE3 equally in the absence and presence of thapsigargin. It was determined using back phosphorylation whether CaM kinase II altered NHE3 phosphorylation. KN-62 led to decreased NHE3 phosphorylation indicating that CaM kinase increased NHE3 phosphorylation. In summary: 1) NHE3 is a CaM kinase II binding protein. 2) CaM kinase II i) binds to NHE3 under basal condition, at a.a. 585 to 605, ii) phosphorylated NHE3 inhibits NHE3 under basal condition. CaM kinase is a regulator of basal NHE3 just as occurs in ileal BB and is part of physiologic NHE3 regulation

NHE3; regulation; calcium/calmodulin dependent protein kinase II

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