EMBO J. 2003 Apr 1;22(7):1478-87.
Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.
van de Graaf, S. F., Hoenderop, J. G., Gkika, D., Lamers, D., Prenen, J., Rescher, U., Gerke, V., Staub, O., Nilius, B., Bindels, R. J.,
["Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands."]
["Department of Cell Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands."]
TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex. Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.
PMID: 12660155

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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 | |||
TRPV5 |
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S100A10 | Yeast two-hybrid | Mouse | C-terminus | Mouse | Kidney | cDNA library | 12660155 | |
TRPV6 |
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S100A10 | Inference | Prediction | 12660155 |
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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 | ||
TRPV5 |
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S100A10 | Fusion protein-pull down assay | Mouse | Full-length | E. coli | Mouse | Full-length | Xenopus oocyte lysates | 12660155 |
TRPV6 |
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S100A10 | Fusion protein-pull down assay | Mouse | Full-length | E. coli | Mouse | Full-length | Xenopus oocyte lysates | 12660155 |
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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 | ||||
TRPV5 |
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S100A10 | Co-immunofluorescence staining | Rat duodenum tissue | 12660155 | |||||
TRPV6 |
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S100A10 | Co-immunofluorescence staining | Rat duodenum tissue | 12660155 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPV5 |
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S100A10 | Fusion protein-pull down assay | Mouse | 591-595 | Mouse | Not determined | 12660155 | ||
TRPV6 |
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S100A10 | Fusion protein-pull down assay | Mouse | 597-601 | Mouse | Not determined | 12660155 |
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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 | ||||||
TRPV5 |
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S100A10 | Co-immunofluorescence staining | Increase in plasma membrane level | 12660155 | |||||||
TRPV5 |
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S100A10 | Patch clamp | Activation | 12660155 | |||||||
TRPV6 |
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S100A10 | Co-immunofluorescence staining | Increase in plasma membrane level | 12660155 | |||||||
TRPV6 |
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S100A10 | Patch clamp | Activation | 12660155 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
