J Biol Chem. 2004 Jun 18;279(25):26351-7. Epub 2004 Apr 20.
80K-H as a new Ca2+ sensor regulating the activity of the epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5).
Gkika, D., Mahieu, F., Nilius, B., Hoenderop, J. G., Bindels, R. J.,
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands."]
["Department of Physiology, Nijmegen Centre for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands."]
The epithelial Ca(2+) channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca(2+) entry pathway in the process of active Ca(2+) reabsorption. Ca(2+) influx through TRPV5 is tightly controlled by modulators of Ca(2+) homeostasis, including 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca(2+)-dependent control of the epithelial Ca(2+) channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). Furthermore, 80K-H directly bound Ca(2+), and inactivation of its two EF-hand structures totally abolished Ca(2+) binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca(2+) current and increased the TRPV5 sensitivity to intracellular Ca(2+), accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca(2+) sensor controlling TRPV5 channel activity.
PMID: 15100231

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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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80K-H | Inference | Prediction | 15100231 |
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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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80K-H | Fusion protein-pull down assay | Mouse | C-terminus | E. coli | Mouse | Full-length | In vitro translation | 15100231 |
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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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80K-H | Co-immunoprecipitation | Xenopus oocyte | Mouse | Full-length | Mouse | Full-length | 15100231 | |
TRPV5 |
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80K-H | Co-immunofluorescence staining | Mouse kidney section | 15100231 |
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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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80K-H | Fusion protein-pull down assay | Mouse | 598-608 | Mouse | Not determined | 15100231 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
