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First published online 15 February 2005
doi: 10.1242/jcs.01675

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Homo- and heteromeric assembly of TRPV channel subunits

Institut für Pharmakologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Thielallee 67-73, 14195 Berlin, Germany

View larger version (57K): [in a new window]   Fig. 1. Expression and subcellular localization of TRPV channels. Rat TRPV1 (A) and murine TRPV2 to TRPV6 (B-F) were C-terminally fused to YFP, and plasmids were transiently transfected in HEK293 cells. Cells were imaged by confocal laser-scanning microscopy 1 day after transfection. The pinholes were adjusted to obtain optical sections with a thickness of 0.6 µm. Typical expression patterns of the different TRPV channels from three to five independent transfections are shown. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 2. Subcellular localization of coexpressed TRPV channel subunits. (A-I) Different combinations of TRPV channels tagged with either CFP or YFP were coexpressed in HEK293 cells and sequentially imaged by confocal laser-scanning microscopy in the same cell. For each coexpression experiment, the correlation coefficient between CFP and YFP fluorescence intensities (r2) was estimated by pixel-based image analysis. Shown are representative data of three independent transfections. Bars, 10 µm.

View larger version (24K): [in a new window]   Fig. 3. Determination of FRET between TRPV channel subunits. (A-D) HEK293 cells were transiently co-transfected with expression plasmids encoding CFP- or YFP-fused TRPV channels as indicated. The relative CFP and YFP fluorescence intensities in single cells expressing the respective TRPV construct were determined and averaged ([CFP]r and [YFP]r) to ensure comparable TRPV expression. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. Cells were excited at 410 nm and 515 nm for CFP and YFP detection, respectively. YFP was bleached with an illumination at 512 nm for 2.1 seconds. FRET efficiencies between identical TRPV channel subunits are shown as grey bars. FRET experiments between different channel subunits are shown as black bars. Bars represent mean±s.e. of at least three independent experiments.

View larger version (29K): [in a new window]   Fig. 4. Coimmunoprecipitation of TRPV channel subunits. (A-E) Plasmids encoding FLAG-tagged or YFP-tagged TRPV channels were co-transfected in HEK293 cells in the combinations indicated below the panels. One day after transfection, membranes were solubilized and lysates were immunoprecipitated (IP) with anti-FLAG antibodies. Membrane lysates (upper panels) or immunoprecipitates (middle and lower panels) were separated by SDS-PAGE. Upper and middle panels, TRPV in the membrane lysates and coprecipitated TRPV channels were detected by immunoblotting (IB) with anti-GFP antibodies. The recovery of the respective immunoprecipitated TRPV-FLAG channel is shown in the lower panels by probing blots with anti-FLAG antibodies. Arrowheads indicate the expected sizes of the respective TRPV-FLAG channel subunits.

View larger version (24K): [in a new window]   Fig. 5. Cytosolic termini are required for TRPV1 assembly and function. Schematic representation of N-terminal (A) and C-terminal (B) TRPV1 deletion mutants showing the ankyrin repeats (indicated as A) and the six transmembrane domains (1-6). WT represents full-length rat TRPV1. The numbers on the left indicate deleted amino acids. The table summarizes the data on the intracellular location of C-terminally YFP-tagged deletion mutants imaged by confocal laser-scanning microscopy, of the capsaicin-induced maximal increases in the intracellular Ca2+ concentrations ([Ca2+]i), and of FRET efficiencies between mutants and full-length TRPV1 (A) or of FRET efficiencies in competition experiments with CFP- and YFP-tagged TRPV1 and non-tagged deletion mutants (B). [YFP]r and [CFP]r represent averaged relative fluorescence intensities of YFP-tagged TRPV1 or the N-terminal TRPV1 deletion mutants and TRPV1-CFP. Data are the mean±s.e. of four to eleven independent FRET experiments. ER, endoplasmic reticulum; PM, plasma membrane.

View larger version (23K): [in a new window]   Fig. 6. Assembly of chimeric TRPV channel subunits. (A) Chimeric TRPV channel subunits were constructed consisting of various combinations of the cytosolic N- or C-termini and the transmembrane domains of TRPV1 and TRPV4. (B-D) HEK293 cells were transiently co-transfected with expression plasmids encoding CFP- or YFP-fused TRPV channels or TRPV channel chimeras as indicated. FRET efficiencies (means±s.e.) were determined as described in Fig. 3. Grey bars, FRET efficiencies of only full-length TRPV channel subunits; black bars, FRET experiments with TRPV channel chimeras. [YFP]r and [CFP]r represent means of the CFP and YFP fluorescence intensities.

View larger version (23K): [in a new window]   Fig. 7. Interaction between cytosolic TRPV1 and TRPV4 termini. (A,B) Cytosolic N- and C-termini (NT, CT) of TRPV1 (A) or TRPV4 (B) tagged with either CFP or YFP were coexpressed in HEK293 cells as indicated and subjected to quantitative FRET analysis. Bars in panels A and B represent FRET efficiencies (means±s.e.) among TRPV termini (grey bars), TRPV termini and full-length TRPV channels (black bars) and, as a control, between TRPV termini and soluble CFP (white bars). Data shown are representative of at least three independent experiments. The average relative fluorescence intensities ([YFP]r and [CFP]r) indicate the relative expression levels of the respective TRPV constructs. (Insets) Confocal images showing typical localization of the YFP-tagged TRPV termini in HEK293 cells (bars, 10 µm).

View larger version (36K): [in a new window]   Fig. 8. Functional rescue of truncated TRPV1 or TRPV4 subunits by fusion with N-termini of related TRPV subunits. (A-C) Plasmids encoding YFP-tagged TRPV chimeras were transfected in HEK293 cells. In the presence of Mn2+ (250 µM), the cells were stimulated by adding 10 µM capsaicin (caps) or 5 µM 4-phorbol 12,13-didecanoate (PDD) to the bath solution as indicated. Grey lines depict the time course of the total fura-2 fluorescence in single cells whereas the black lines represent the calculated means. The data are representative for three to four independent transfection experiments showing similar results. Insets show confocal micrographs of living HEK293 cells expressing the respective YFP-tagged TRPV chimera. Bar, 10 µm.

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