Cell Calcium. 2008 Mar;43(3):260-9. Epub 2007 Jul 12.
The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels.
Schindl, R., Frischauf, I., Kahr, H., Fritsch, R., Krenn, M., Derndl, A., Vales, E., Muik, M., Derler, I., Groschner, K., Romanin, C.,
["Institute for Biophysics, University of Linz, A-4040 Linz, Austria."]
["Institute for Biophysics, University of Linz, A-4040 Linz, Austria."]
The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Forster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.
PMID: 17624425

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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 | ||||
TRPC4 |
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TRPC5 | Fluorescence resonance energy transfer | HEK293 | Mouse | Full-length | Mouse | Full-length | 17624425 | |
TRPC5 |
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TRPC4 | Fluorescence resonance energy transfer | HEK293 | Mouse | Full-length | Mouse | Full-length | 17624425 |
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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 | ||||
TRPC4 |
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TRPC5 | Fluorescence resonance energy transfer | Mouse | 1-292 | Mouse | 1-308 | 17624425 | ||
TRPC5 |
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TRPC4 | Fluorescence resonance energy transfer | Mouse | 1-308 | Mouse | 1-292 | 17624425 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
