title: Distinct modes of perimembrane TRP channel turnover revealed by TIR-FRAP.
abstract: Transient Receptor Potential (TRP) channels form a broadly expressed and functionally diverse family of cation channels involved in various (patho)physiological processes. Whereas the mechanisms that control opening of TRP channels have been extensively studied, little is known about the transport processes of TRP channels to and within the plasma membrane. Here we used Total Internal Reflection--Fluorescence Recovery after Photobleaching (TIR-FRAP) to selectively visualize and bleach the fluorescently labeled TRP channels TRPV2 and TRPM4 in close proximity of the glass-plasma membrane interface, allowing detailed analysis of their perimembrane dynamics. We show that recovery of TRPM4 occurs via 200-nm diameter transport vesicles, and demonstrate the full fusion of such vesicles with the plasma membrane. In contrast, TRPV2 recovery proceeded mainly via lateral diffusion from non-bleached areas of the plasma membrane. Analysis of the two-dimensional channel diffusion kinetics yielded 2D diffusion coefficients ranging between 0.1 and 0.3 μm(2)/s, suggesting that these TRP channels move relatively unrestricted within the plasma membrane. These data demonstrate distinct modes of TRP channel turnover at the plasma membrane and illustrate the usefulness of TIR-FRAP to monitor these processes with high resolution.
authors.affiliation: Laboratory of Ion Channel Research and TRP channel Research Platform Leuven (TRPLe) Department of Cellular and Molecular Medicine, KU Leuven, Belgium.
authors.forename: Debapriya
authors.initials: D
authors.lastname: Ghosh
authors.affiliation: Laboratory of Ion Channel Research and TRP channel Research Platform Leuven (TRPLe) Department of Cellular and Molecular Medicine, KU Leuven, Belgium.
authors.forename: Andrei
authors.initials: A
authors.lastname: Segal
authors.affiliation: Laboratory of Ion Channel Research and TRP channel Research Platform Leuven (TRPLe) Department of Cellular and Molecular Medicine, KU Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
mesh_terms: D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human; D050916:TRPV Cation Channels; C400769:TRPV2 protein, human; D049452:Green Fluorescent Proteins