Biochem Biophys Res Commun. 2007 Sep 21;361(2):433-8. Epub 2007 Jul 16.

PDZ domain-containing protein as a physiological modulator of TRPV6.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Kim, H. J., Yang, D. K., So, I.,
["Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea."]
The epithelial Ca(2+) channel TRPV6 constitutes the apical Ca(2+) entry mechanism in active Ca(2+) transport in kidney and intestine, but little is known about regulation mechanism of TRPV6. We performed yeast two-hybrid screen with TRPV6 C-terminus since TRPV6 has PDZ (Post-synaptic density-95, Drosophila discs-large protein, Zonula occludens protein 1) binding motif at its C terminal end. As a result, we found that 4 PDZ domain-containing protein, PDZK2, interacts with TRPV6 through its fourth PDZ domain. Glutathione S-transferase pull-down assay shows that TRPV6 and PDZK2 directly interact and that TRPV6 C-terminal PDZ binding motif is primarily responsible for this interaction. Mutant Delta4 lacking last 4 amino acid EYQI did not interact with PDZK2. Heterologous overexpression of both TRPV6 and PDZK2 did not show any effect on the activation of TRPV6. On the other hand, the peak current amplitude of mutant Delta4 decreased compared with that of WT TRPV6. When introduced into HEK293 cells expressing TRPV6, PDZ binding motif peptide (EYQI) markedly reduced the peak current amplitude in divalent free (DVF) solution. Knocking down the endogenous PDZK2 of HEK293 cells by RNAi significantly decreased DVF current density. Taken together, we propose that PDZK2 is an essential TRPV6 interacting protein as a physiological modulator of TRPV6.
PMID: 17645868External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Yeast two-hybrid Mouse 575-727 Mouse Kidney cDNA library 17645868
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse 575-727 E. coli Mouse Full-length In vitro translation 17645868
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Fusion protein-pull down assay Mouse 724-727 Mouse Not determined 17645868
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPV6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF4 Patch clamp Activation 17645868
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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