title: Ca2+-selective transient receptor potential V channel architecture and function require a specific ankyrin repeat.
abstract: Transient receptor potential (TRP) proteins form cation-conducting ion channels with currently 28 known genes encoding TRP channel monomers in mammals. These monomers are thought to coassemble to form homo- or heterotetrameric channels, but the signals governing their assembly are unknown. Within the TRPV subgroup, TRPV5 and TRPV6 show exclusive calcium selectivity and play an important role in calcium uptake. To identify signals that mediate assembly of functional TRPV6, we screened domains for self-association using co-immunoprecipitation, sucrose gradient centrifugation, bacterial two-hybrid assays, and patch clamp analysis. Of the two identified interaction domains within the N-terminal region, we showed that the first domain encompassing the third ankyrin repeat is the stringent requirement for physical assembly of TRPV6 subunits and when transferred to an unrelated protein enables its interaction with TRPV6. Deletion of this repeat or mutation of critical residues within this repeat rendered nonfunctional channels that do not co-immunoprecipitate or form tetramers. Suppression of dominant-negative inhibitors of TRPV6-specific currents was achieved by deletion of ankyrin (ANK) 3. We propose that the third ANK repeat initiates a molecular zippering process that proceeds past the fifth ANK repeat and creates an intracellular anchor that is necessary for functional subunit assembly.
authors.affiliation: Experimentelle und Klinische Pharmakologie und Toxikologie der Universität des Saarlandes, 66421 Homburg, Germany.
authors.forename: Isabell
authors.initials: I
authors.lastname: Erler
authors.forename: Daniela
authors.initials: D
authors.lastname: Hirnet
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wissenbach
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Niemeyer
mesh_terms: D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry*; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000502:physiology; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D017353:Gene Deletion; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006801:Humans; D007477:Ions; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D018408:Patch-Clamp Techniques; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D050916:TRPV Cation Channels; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D017487:Ankyrins; D015220:Calcium Channels; D007477:Ions; D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C120776:TRPV6 channel; D049452:Green Fluorescent Proteins; D013395:Sucrose; D002118:Calcium