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Related Experiment Videos

Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma

Whaseon Lee-Kwon1, Jae Ho Kim, Jung Woong Choi

  • 1Johns Hopkins Univ. School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195, USA.

American Journal of Physiology. Cell Physiology
|September 5, 2003
PubMed
Summary
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Elevated intracellular calcium inhibits the intestinal brush border Na+/H+ exchanger 3 (NHE3) via protein kinase C (PKC)alpha, which is necessary for decreasing NHE3 membrane levels, likely through endocytosis.

Area of Science:

  • Cell Biology
  • Physiology
  • Molecular Biology

Background:

  • The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) plays a crucial role in ion absorption.
  • Intracellular calcium ([Ca2+]i) regulates NHE3 activity in a protein kinase C (PKC)-dependent manner.
  • The PDZ domain-containing protein E3KARP is involved in the Ca2+-dependent regulation of NHE3.

Purpose of the Study:

  • To investigate the precise mechanism of PKC involvement in Ca2+ regulation of NHE3.
  • To elucidate the role of PKCalpha in the Ca2+-induced inhibition of NHE3 activity and membrane trafficking.
  • To determine the interaction between PKCalpha, E3KARP, and NHE3.

Main Methods:

  • Utilized PS120 fibroblasts stably transfected with NHE3 and E3KARP.

Related Experiment Videos

  • Employed PKC inhibitors (GF109203X, Gö-6976) and a specific PKCalpha inhibitor peptide.
  • Assessed protein translocation via Western blotting and co-immunoprecipitation.
  • Investigated NHE3 oligomerization and plasma membrane abundance using biotinylation assays.

    • Main Results:

    • PKC inhibitors blocked ionomycin-induced NHE3 inhibition without affecting basal activity.
    • [Ca2+]i elevation led to PKCalpha translocation to the membrane and binding to E3KARP.
    • PKCalpha and E3KARP co-immunoprecipitated, with increased interaction upon ionomycin exposure.
    • While Ca2+ induced NHE3 oligomerization, PKC inhibitors prevented the decrease in plasma membrane NHE3, but not oligomerization.

    Conclusions:

    • PKCalpha is essential for the Ca2+-dependent decrease in plasma membrane NHE3 abundance.
    • PKCalpha likely stimulates NHE3 endocytosis, leading to its removal from the plasma membrane.
    • PKCalpha is not required for the Ca2+-dependent formation of the NHE3-E3KARP complex but is critical for subsequent trafficking events.