J Biol Chem. 2004 Jul 9;279(28):28855-61. Epub 2004 Apr 30.
Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin.
Lambers, T. T., Weidema, A. F., Nilius, B., Hoenderop, J. G., Bindels, R. J.,
["Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands."]
["Department of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Centre Nijmegen, NL-6500 HB Nijmegen, The Netherlands."]
TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) as well as the Na(+) current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca(2+)-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini (1-8-14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.
PMID: 15123711

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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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Calmodulin | Inference | Prediction | 15123711 |
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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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Calmodulin | Fusion protein-pull down assay | Mouse | Full-length | In vitro translation | Not specified | Full-length | Not specified | 15123711 |
TRPV5 |
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Calmodulin | Fusion protein-pull down assay | Mouse | 1-327 | In vitro translation | Not specified | Full-length | Not specified | 15123711 |
TRPV5 |
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Calmodulin | Fusion protein-pull down assay | Mouse | 578-730 | In vitro translation | Not specified | Full-length | Not specified | 15123711 |
TRPV6 |
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Calmodulin | Fusion protein-pull down assay | Rabbit | Full-length | In vitro translation | Not specified | Full-length | Not specified | 15123711 |
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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 | ||||
TRPV6 |
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Calmodulin | Co-immunoprecipitation | Xenopus oocyte | Rabbit | Full-length | Not specified | Full-length | 15123711 |
(
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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 | ||||
TRPV6 |
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Calmodulin | Fusion protein-pull down assay | Rabbit | 327-577 | Not specified | Not determined | 15123711 | ||
TRPV6 |
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Calmodulin | Fusion protein-pull down assay | Rabbit | 88-97 | Not specified | Not determined | 15123711 | ||
TRPV6 |
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Calmodulin | Fusion protein-pull down assay | Rabbit | 643-656 | Not specified | Not determined | 15123711 |
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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 | ||||||
TRPV6 |
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Calmodulin | Patch clamp | Inhibition | 15123711 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
