Glia. 2005 Feb;49(3):418-29.

Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Song, X., Zhao, Y., Narcisse, L., Duffy, H., Kress, Y., Lee, S., Brosnan, C. F.,
["Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA."]
Members of the mammalian transient receptor potential (TRP) family form cation-permeable channels at the plasma membrane implicated in capacitative calcium influx after activation by either second-messenger-mediated pathways or store depletion, or both. This study shows that with the use of RT-PCR, Western blotting, and immunohistochemistry, resting astrocytes express TRPC4 at the cell membrane, particularly at sites of cell-to-cell contact. By confocal imaging and immunoelectron microscopy, we detected co-localization of TRPC4 with the scaffolding protein zonula occludens 1 (ZO-1), and demonstrated that immunoprecipitation with antibodies to ZO-1 brought down TRPC4, and vice-versa. It has been proposed that the targeting of TRPC4 to the cell membrane is dependent on the interaction of the C-terminal TRL motif with PDZ domains. Using transfection of astrocytes with myc-tagged TRPC4 or TRL-motif truncated TRPC4 (deltaTRL), we found that deltaTRL localized predominantly to a juxtanuclear compartment, whereas the wild-type protein showed cell surface distribution. Deletion of the TRL motif also reduced plasma membrane expression as assessed by cell surface biotinylation experiments. Using GST fusion proteins, we found that TRPC4 interacted with the PDZ1 domain of ZO-1 and that this was also dependent on the TRL motif. Thus, our data demonstrate that the PDZ-interacting domain of TRPC4 controls its cell surface localization. These data implicate TRPC4 in the regulation of calcium homeostasis in astrocytes, particularly as part of a signaling complex that forms at junctional sites between astrocytes.
PMID: 15540229External 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
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GJA1 Inference Prediction 15540229
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ZO-1 Inference Prediction 15540229
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GJA1 Co-immunofluorescence staining Human fetal brain astrocytes 15540229
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 GJA1 Co-immunoprecipitation Human fetal brain astrocytes 15540229
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF1 Co-immunoprecipitation Human fetal brain astrocytes 15540229
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ZO-1 Co-immunofluorescence staining Human fetal brain astrocytes 15540229
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ZO-1 Co-immunoprecipitation Human fetal brain astrocytes 15540229
(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
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ZO-1 Fusion protein-pull down assay Human 890-892 Not specified First PDZ domain 15540229
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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