J Biol Chem. 2006 Oct 27;281(43):32540-9. Epub 2006 Aug 3.
Homer 1 mediates store- and inositol 1,4,5-trisphosphate receptor-dependent translocation and retrieval of TRPC3 to the plasma membrane.
Kim, J. Y., Zeng, W., Kiselyov, K., Yuan, J. P., Dehoff, M. H., Mikoshiba, K., Worley, P. F., Muallem, S.,
["Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA."]
["Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA."]
Store-operated Ca(2+) channels (SOCs) mediate receptor-stimulated Ca(2+) influx. Accumulating evidence indicates that members of the transient receptor potential (TRP) channel family are components of SOCs in mammalian cells. Agonist stimulation activates SOCs and TRP channels directly and by inducing translocation of channels in intracellular vesicles to the plasma membrane (PM). The mechanism of TRP channel translocation in response to store depletion and agonist stimulation is not known. Here we use TRPC3 as a model to show that IP(3) and the scaffold Homer 1 (H1) regulate the rate of translocation and retrieval of TRPC3 from the PM. In resting cells, TRPC3 exists in TRPC3-H1b/c-IP(3)Rs complexes that are located in part at the PM and in part in intracellular vesicles. Binding of IP(3) to the IP(3)Rs dissociates the interaction between IP(3)Rs and H1 but not between H1 and TRPC3 to form IP(3)Rs-TRPC3-H1b/c. TIRFM and biotinylation assays show robust receptor- and store-dependent translocation of the TRPC3 to the PM and their retrieval upon termination of cell stimulation. The translocation requires depletion of stored Ca(2+) and is prevented by inhibition of the IP(3)Rs. In HEK293, dissociating the H1b/c-IP(3)R complex with H1a results in TRPC3 translocation to the PM, where it is spontaneously active. The TRPC3-H1b/c-IP(3)Rs complex is reconstituted by infusing H1c into these cells. Reconstitution is inhibited by IP(3). Deletion of H1 in mice markedly reduces the rates of translocation and retrieval of TRPC3. Conversely, infusion of H1c into H1(-/-) cells eliminates spontaneous channel activity and increases the rate of channel activation by agonist stimulation. The effects of H1c are inhibited by IP(3). These findings together with our earlier studies demonstrating gating of TRPC3 by IP(3)Rs were used to develop a model in which assembly of the TRPC3-H1b/c-IP(3)Rs complexes by H1b/c mediates both the translocation of TRPC3-containing vesicles to the PM and gating of TRPC3 by IP(3)Rs.
PMID: 16887806

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC3 |
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Homer-1 | Inference | Prediction | 16887806 | |||||
TRPC3 |
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PMCA | Speculation | Prediction | 16887806 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPC3 |
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Homer-1 | Fusion protein-pull down assay | Human | Full-length | HEK293 lysates | Human | Full-length | E. coli | 16887806 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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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 | ||||
TRPC3 |
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IP3R3 | Co-immunofluorescence staining | Mouse pancreatic acini | 16887806 | |||||
TRPC3 |
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IP3R3 | Co-immunofluorescence staining | Mouse parotid acini | 16887806 | |||||
TRPC3 |
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IP3R3 | Co-immunofluorescence staining | Mouse submandibular gland duct | 16887806 | |||||
TRPC3 |
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IP3R3 | Co-immunoprecipitation | Mouse brain tissue | 16887806 | |||||
TRPC3 |
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IP3R3 | Co-immunoprecipitation | Mouse pancreatic tissue | 16887806 | |||||
TRPC3 |
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IP3R3 | Co-immunoprecipitation | Mouse submandibular gland lysates | 16887806 | |||||
TRPC3 |
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PMCA | Co-immunoprecipitation | Mouse brain tissue | 16887806 | |||||
TRPC3 |
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PMCA | Co-immunoprecipitation | Mouse pancreatic tissue | 16887806 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPC3 |
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Homer-1 | Patch clamp | Activation | 16887806 | |||||||
TRPC3 |
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Homer-1 | Cell surface biotinylation | Increase in plasma membrane level | 16887806 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
