Ca2+-dependent inhibition of NHE3 requires PKCα which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes

Whaseon Lee-Kwon, Jae Ho Kim, Jung Woong Choi, Kazuya Kawano, Boyoung Cha, Darlene A. Dartt, Driss Zoukhri, Mark Donowitz

Research output: Contribution to journalArticlepeer-review

83 Scopus citations


The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca 2+]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 Ca 2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCα pseudosubstrate-derived inhibitor peptide. [Ca2+] i elevation caused translocation of PKCα from cytosol to membrane. PKCα bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCα and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca 2+]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 PKCα is not necessary for the Ca 2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.

Original languageEnglish (US)
Pages (from-to)C1527-C1536
JournalAmerican Journal of Physiology - Cell Physiology
Issue number6 54-6
StatePublished - Dec 2003
Externally publishedYes


  • Na absorption
  • PDZ domains
  • Signal complex

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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