title: Intracellular coiled-coil domain engaged in subunit interaction and assembly of melastatin-related transient receptor potential channel 2.
abstract: TRPM2 channels, activated by adenosine diphosphoribose and related molecules, are assembled as oligomers and most likely tetramers. However, the molecular determinants driving the subunit interaction and assembly of the TRPM2 channels are not well defined. Here we examined, using site-directed mutagenesis in conjunction with co-immunoprecipitation and patch clamp recording, the role of a coiled-coil domain in the intracellular C terminus of TRPM2 subunit in subunit interaction and channel assembly. Deletion of the coiled-coil domain resulted in severe disruption of the subunit interaction and substantial loss of the adenosine diphosphoribose-evoked channel currents. Individual or combined mutations to glutamine of the hydrophobic residues at positions a and d of the abcdef heptad repeat, key residues for protein-protein interaction, significantly reduced the subunit interaction and channel currents; the mutational effects on the subunit interaction and channel currents were clearly correlated. Furthermore, deletion of the coiled-coil domain in a pore mutant subunit abolished its dominant negative phenotypic functional suppression. These results provide strong evidence that the coiled-coil domain is critically engaged in the TRPM2 subunit interaction and such interaction is required for assembly of functional TRPM2 channel. The coiled-coil domain, which is highly conserved within the TRPM subfamily, may serve as a general structural element governing the assembly of TRPM channels.
authors.affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Zhu-Zhong
authors.initials: ZZ
authors.lastname: Mei
authors.forename: Rong
authors.initials: R
authors.lastname: Xia
authors.forename: David J
authors.initials: DJ
authors.lastname: Beech
authors.forename: Lin-Hua
authors.initials: LH
authors.lastname: Jiang
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D050053:TRPM Cation Channels / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D050053:TRPM Cation Channels; C492059:TRPM1 protein, human; C121934:TRPM2 protein, human