title: The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels.
abstract: 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 Förster 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.
authors.affiliation: Institute for Biophysics, University of Linz, A-4040 Linz, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: Schindl
authors.forename: Irene
authors.initials: I
authors.lastname: Frischauf
authors.forename: Heike
authors.initials: H
authors.lastname: Kahr
authors.forename: Reinhard
authors.initials: R
authors.lastname: Fritsch
authors.forename: Martin
authors.initials: M
authors.lastname: Krenn
authors.forename: Alexandra
authors.initials: A
authors.lastname: Derndl
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Vales
authors.forename: Martin
authors.initials: M
authors.lastname: Muik
authors.forename: Isabella
authors.initials: I
authors.lastname: Derler
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat; D002460:Cell Line; D005799:Genes, Dominant; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013329:Structure-Activity Relationship; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D010446:Peptide Fragments; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C112021:Trpc5 protein, mouse