Pflügers Archiv

, Volume 452, Issue 4, pp 407–417 | Cite as

Interaction of the epithelial Ca2+ channels TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ protein NHERF4

  • Stan F. J. van de Graaf
  • Joost G. J. Hoenderop
  • Annemiete W. C. M. van der Kemp
  • Serge M. Gisler
  • René J. M. BindelsEmail author
Cell and Molecular Physiology


The epithelial Ca2+ channels TRPV5 and TRPV6 constitute the apical Ca2+ influx pathway in epithelial Ca2+ transport. PDZ proteins have been demonstrated to play a crucial role in the targeting or anchoring of ion channels and transporters in the apical domain of the cell. In this study, we describe the identification of NHERF4 (Na-Pi Cap2/IKEPP/PDZK2) as a novel TRPV5- and TRPV6-associated PDZ protein. NHERF4 was identified using two separate yeast two-hybrid screens with the carboxyl termini of TRPV5 and TRPV6 as bait. Binding of the carboxyl termini of TRPV5 and TRPV6 with NHERF4 was confirmed by GST pull-down assays using in-vitro-translated NHERF4 or lysates of Xenopus laevis oocytes expressing NHERF4. Furthermore, the interaction was confirmed by GST pull-down and co-immunoprecipitation assays using in-vitro-translated full-length TRPV5 and Xenopus oocytes or HEK293 cells co-expressing NHERF4 and TRPV5/TRPV6, respectively. The fourth PDZ domain of NHERF4 was sufficient for the interaction, although PDZ domain 1 also contributed to the binding. The binding site for NHERF4 localized in a conserved region in the carboxyl terminus of TRPV5 and was distinct from the binding site of the PDZ protein NHERF2. NHERF4 predominantly localized at the plasma membrane of X. laevis oocytes and HeLa cells. This localization was independent of the presence of TRPV5. Therefore, we hypothesize a role for this novel PDZ protein as a putative plasma membrane scaffold for the epithelial Ca2+ channels.


ECaC1 CaT1 Calcium homeostasis PDZ Protein–protein interactions 



The authors thank Dr. Arjen Mensenkamp for the TRPV4 construct. This work was sponsored by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298, NWO-ALW 810.38.004, NWO-ALW 805.09.042, NWO-ALW 814.02.001, and NWO-Talent S91-282).


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Stan F. J. van de Graaf
    • 1
    • 3
  • Joost G. J. Hoenderop
    • 1
  • Annemiete W. C. M. van der Kemp
    • 1
  • Serge M. Gisler
    • 2
  • René J. M. Bindels
    • 1
    Email author
  1. 1.Cell Physiology, Department of Physiology (286), Nijmegen Center for Molecular Life SciencesRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
  2. 2.Institute of PhysiologyUniversity of Zürich-IrchelZürich-IrchelSwitzerland
  3. 3.Institute of Medical Biochemistry, Center for Molecular Biology of InflammationUniversity of MuensterMuensterGermany

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