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Am J Physiol Cell Physiol (September 3, 2003). doi:10.1152/ajpcell.00017.2003
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Submitted on January 14, 2003
Accepted on August 8, 2003

Ca2+-Dependent Inhibition of NHE3 Requires PKC{alpha} Which Binds to E3KARP to Decrease Surface NHE3 Containing Plasma Membrane Complexes

Whasseon Lee-Kwon1, Jae Ho Kim2, Jung Woong Choi1, Kazuya Kawano1, Boyoung Cha1, Darlene A Dartt3, Dris Zoukhri3, and Mark Donowitz1*

1 Medicine/Physiology, Johns Hopkins University School of Medicine, Balt, MD, USA
2 Medicine/Physiology, Johns Hopkins University School of Medicine, Balt, MD, USA; Physiology, Pusan National University, Pusan, Korea, Republic of
3 Harvard Medical School and Schepens Eye Research Institute, Boston, MA, USA

* To whom correspondence should be addressed. E-mail: mdonowit{at}Jhmi.edu.

The intestinal brush border (BB) Na+/H+ Exchanger Isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([CaM2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC) dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+ mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Go6976 and a specific PKC{alpha} pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKC{alpha} from cytosol to membrane. PKC{alpha} bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+ dependent manner. PKC{alpha} and E3KARP co-immunoprecipitated from cell lysates; this occurred to a small extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC{alpha} is not necessary for the Ca2+ dependent formation of the NHE3 plasma membrane complex, while it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.




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