Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Physical and functional coupling of native TRPC5 proteins associated with eNOS in BAECs. A , [Ca 2+ ] i increases (340:380-nm fluorescence ratio; F340/F380) in sieNOS- or siControl-transfected BAECs in response to ATP. Representative time courses ( left ) and averaged [Ca 2+ ] i increases during 480 to 600 s ( right ) ( n = 64 each). The averaged ratio during 10 to 50 s was subtracted as background for each of the cells to derive ΔF340/F380. B , intracellular NO production evoked by 1 μM ATP in BAECs treated with or without 10 μM L-NAME. Representative time courses of DAF-2 fluorescent changes (F/F 0 ) ( left ) and maximal fluorescent increases (ΔF/F 0 ) ( right ) ( n = 19–38). C , [Ca 2+ ] i increases in sieNOS- or siControl-transfected BAECs in 0.5 mM EGTA (Ca 2+ -free)- or 2 mM Ca 2+ -containing solution. Representative time courses ( left ) and averaged [Ca 2+ ] i increases during 980–1020 s ( right ) ( n = 20–25). In A to C , 3 mM L-arginine is added to the bath solutions. D , confocal fluorescence images of protein localization in BAECs. Specific antibodies are used to detect TRPC5 ( green ) and eNOS ( red ). The nuclei are stained with Hoechst 33342 ( blue ). The bar indicates 10 μm. E , confocal fluorescence images of TRPC5-eNOS colocalization in BAEC visualized by proximity ligation assay (PLA). Specific antibodies for TRPC5 and eNOS were used to obtain PLA signal ( red ). The nuclei are stained with DAPI ( blue ). The bar indicates 5 μm. F, co-immunoprecipitation of TRPC5 with eNOS in BAEC. Immunoprecipitates (IPs) with or without anti-eNOS antibody are subjected to western blotting (WB) with antibody to TRPC5 ( left ). Right , quantification of gel image. G , [Ca 2+ ] i increases evoked by 1 μM ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in siTRPC5- or siControl-transfected BAECs treated with or without 10 μM L-NAME. Representative time courses ( left ) and averaged [Ca 2+ ] i increases (ΔF340/F380) during 980–1020 s ( right ) ( n = 38–78). p values are determined by one-way ANOVA with subsequent paired comparison. H , S -nitrosylation assay (biotin switch assay) of TRPC5 in BAECs. The cells are treated with 1 μM ATP for 5 min. The collected cell lysates are treated with biotin-HPDP to selectively label S -nitrosylated proteins ( left ). Right , quantification of gel image. For A , B , C , and G , individual data points indicate individual cells examined, and for all the gel images, individual data points indicate technical replicates. Data points are mean ± SD.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Functional Assay, Fluorescence, Transfection, Staining, Proximity Ligation Assay, Immunoprecipitation, Western Blot, Comparison, Biotin Switch Assay

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: TRPC subtype-selectivity of functional coupling with eNOS in HEK293 cells. A , confocal fluorescent images of HEK293 cells expressing eNOS-GFP and TRPC5-DsRed. The bar indicates 5 μm. The insets show enlarged views of the boxed regions ( left ). The bar indicates 0.5 μm. Right , quantification of the area exhibiting merged signal relative to the area exhibiting TRPC5 signal. B , confocal fluorescence images of TRPC5-eNOS colocalization in HEK293 visualized by PLA. Specific antibodies for TRPC5 and eNOS were used to obtain PLA signal ( red ). The nuclei are stained with DAPI ( blue ). The bar indicates 5 μm. C , [Ca 2+ ] i increases evoked by ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in TRPC5-expressing HEK293 cells cotransfected with vector or eNOS. Averaged time courses ( left ) and dose-response relationships of maximal [Ca 2+ ] i increases in 2 mM Ca 2+ -containing solution ( right ) ( n = 33–34). ∗ p < 0.05 and ∗∗ p < 0.01, as compared to the cells cotransfected with TRPC5 and vector. D , maximal [Ca 2+ ] i increases evoked by 100 μM ATP in 2 mM Ca 2+ -containing solution in TRPC homolog-expressing HEK293 cells cotransfected with vector or eNOS ( n = 13–68). p values determined by two-way ANOVA with subsequent paired comparison. E , co-immunoprecipitation of eNOS-GFP occurs with TRPC5-Flag but not with TRPC3-Flag. IPs with Flag-specific antibody are subjected to WB with antibody to GFP ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. F , [Ca 2+ ] i increases evoked by 100 μM ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in TRPC5- and eNOS-expressing HEK293 cells treated with or without 300 μM L-NAME. Averaged time courses ( left ) and averaged [Ca 2+ ] i increases during 920–1050 s in Ca 2+ -containing solution ( right ) ( n = 42–45). G , DAF-2 fluorescent changes evoked by 1 mM ATP in eNOS-expressing HEK293 cells cotransfected with vector or TRPC5. Averaged time courses (F/F 0 ) ( left ) and averaged fluorescent increases during 1080–1200 s (ΔF/F 0 ) ( right ) ( n = 18–23). In C, D, F , and G , 3 mM L-arginine is added to the bath solutions. H , S -nitrosylation assay in HEK293 cells. Cells cotransfected with TRPC5-GFP and eNOS constructs are treated with or without 100 μM ATP for 10 min. The collected cell lysates are treated with biotin-HPDP to selectively label S -nitrosylated proteins ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. I , representative time courses of 100 μM ATP-evoked outward and inward whole cell currents under ramp clamp conditions, in TRPC5-expressing HEK293 cells cotransfected with eNOS construct. The corresponding I - V relationships at time points 1 and 2 are also shown. J , ATP-evoked current responses ( ΔI ) of TRPC5 at −100 mV (n = 11–17). p values are determined by one-way ANOVA with subsequent paired comparison. Three mM L-arginine is added to the bath solutions. For D , F , G , and J , individual data points indicate individual cells examined, and for all the gel images, individual data points indicate technical replicates. Data points are mean ± SD.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Functional Assay, Expressing, Fluorescence, Staining, Plasmid Preparation, Comparison, Immunoprecipitation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Caveolin-1 binds to the C-terminus of TRPC5 to form a complex. A , confocal fluorescence images of protein localization in BAECs. Specific antibodies are used to detect TRPC5 ( green ) and caveolin-1 proteins ( red ). The nuclei are stained with Hoechst 33342 ( blue ). The bar indicates 10 μm. B , co-immunoprecipitation of TRPC5 with caveolin-1 in BAEC. IPs with anti-caveolin-1 antibody are subjected to WB with antibody to TRPC5 ( left ). Right , quantification of gel image. C , co-immunoprecipitation of TRPC5-GFP with caveolin-1-Flag in HEK293 cells. IPs with Flag-specific antibody are subjected to WB with antibody to GFP ( left ). Right , quantification of gel image. D , a schematic representation of GST fusion proteins of mouse TRPC5 subfragments. E , pull down assay of caveolin-1 with GST fusion proteins of TRPC5 fragments. GST fusion proteins immobilized on glutathione-sepharose beads are incubated with cell lysates obtained from HEK293 cells transfected with caveolin-1-GFP. Bound proteins are analyzed by WB with antibody to GFP ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. F , structural analysis of the caveolin-1–binding region of TRPC5. Red regions represent the putative caveolin-1–binding motif 295 to 322 (PDBID: 7e4t ). G , an alignment of the putative caveolin-1–binding domain in the N-terminus for mouse TRPC proteins. H , co-immunoprecipitation studies of TRPC5-GFP constructs with caveolin-1-Flag. IPs with Flag-specific antibody are subjected to WB with antibody to GFP ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. Individual data points indicate technical replicates for all the gel images.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Fluorescence, Staining, Immunoprecipitation, Pull Down Assay, Incubation, Transfection, Comparison, Binding Assay, Construct

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Th e mutation in the caveolin-1–binding domain of TRPC5 and eNOS abrogates their physical and functional coupling. A , [Ca 2+ ] i increases evoked by 100 μM ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in TRPC5 Y296A/F301A -expressing HEK293 cells cotransfected with eNOS or vector. Dashed line indicates the result obtained from WT TRPC5 cotransfected with eNOS in a separate experiment for comparison. Averaged time courses ( left ) and maximal [Ca 2+ ] i increases in Ca 2+ -containing solution ( right ) ( n = 39–72). p values are determined by one-way ANOVA with subsequent paired comparison. Three millimolars of L-arginine is added to the bath solutions. B , co-immunoprecipitation studies of TRPC5-GFP constructs with eNOS-Flag. IPs with Flag-specific antibody are subjected to WB with antibody to GFP ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. C , confocal fluorescent images of HEK293 cells expressing TRPC5 Y296A/F301A -GFP and eNOS-DsRed. The bar indicates 5 μm. The insets show enlarged views of the boxed regions ( left ). The bar indicates 0.5 μm. Right , quantification of the area exhibiting merged signal relative to the area exhibiting TRPC5 Y296A/F301A signal. D , co-immunoprecipitation studies of eNOS-GFP constructs with caveolin-1-Flag ( left ). Right , quantification of gel image. E , co-immunoprecipitation studies of eNOS-GFP constructs with TRPC5-Flag ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. F, [Ca 2+ ] i increases evoked by 100 μM ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in TRPC5-expressing HEK293 cells cotransfected with eNOS constructs. Averaged time courses ( left ) and maximal [Ca 2+ ] i increases (ΔF340/F380) in EGTA- or Ca 2+ -containing solution ( right ) (n = 40–52). p values are determined by one-way ANOVA with subsequent paired comparison. G, [Ca 2+ ] i increases of siCaveolin-1- or siControl-transfected BAECs in 2 mM Ca 2+ -containing solution evoked by 100 μM ATP. Averaged [Ca 2+ ] i increases (ΔF340/F380) during 980–1020 s are shown (n = 32–33). For A , F , and G , individual data points indicate individual cells examined, and for all the gel images, individual data points indicate technical replicates. Data points are mean ± SD.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Mutagenesis, Binding Assay, Functional Assay, Expressing, Plasmid Preparation, Comparison, Immunoprecipitation, Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Formation of TRPC5–eNOS complexes is enhanced and remodeled by [Ca 2+ ] i elevation during receptor stimulation. A , representative FRET/CFP ratio images (pseudo-color) of HEK293 cells co-expressing TRPC5-CFP and YFP or TRPC5-CFP and eNOS-YFP upon treatment with or without 100 μM ATP in 2 mM Ca 2+ -containing solution. The bars indicate 10 μm. B , averaged time courses ( left ) of the normalized FRET/CFP ratio in the plasma membrane area of HEK293 cells co-expressing TRPC5-CFP with eNOS-YFP, CFP with YFP, or TRPC5-CFP with YFP upon treatment with 100 μM ATP in 2 mM Ca 2+ -containing solution ( n = 7–8). Data are presented as the FRET/CFP ratio normalized to that at t = 0. Averaged increases ( right ) in normalized FRET/CFP ratio during 510 to 600 s ( n = 7–8). p values are determined by one-way ANOVA with subsequent paired comparison. C , co-immunoprecipitation of eNOS-GFP with TRPC5-Flag under treatment with 100 μM ATP for the indicated time (min) in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution. IPs with Flag-specific antibody are subjected to WB with antibody to GFP. D , time courses of the amount of bound proteins, normalized with respect to cell lysate protein concentrations ( n = 3–7). p values are determined by two-way ANOVA with subsequent paired comparison. E , co-immunoprecipitation of eNOS-GFP with TRPC5-Flag under treatment with 100 μM ATP for the indicated time (min) in 2 mM Ca 2+ -containing solution, using cell lysates obtained from HEK293 cells cotransfected with eNOS-GFP, CaM-Mut, and either TRPC5-Flag or Flag ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. F , [Ca 2+ ] i increases evoked by 100 μM ATP in 0.5 mM EGTA- or 2 mM Ca 2+ -containing solution, in TRPC5-expressing HEK293 cells cotransfected with CaM constructs and either eNOS or vector. Averaged time courses ( left ) and maximal [Ca 2+ ] i increases in Ca 2+ -containing solution ( right ) ( n = 35–80). p values are determined by one-way ANOVA with subsequent paired comparison. G and H , pull down assay of eNOS with GST fusion proteins of TRPC5 fragments. GST fusion proteins immobilized on glutathione-Sepharose beads are incubated with cell lysates obtained from HEK293 cells cotransfected with eNOS-GFP and CaM in 100 nM ( G ) or 10 μM ( H ) Ca 2+ -containing solution. Bound proteins are analyzed by WB with antibody to GFP ( left ). Right , quantification of gel image. p values are determined by one-way ANOVA with subsequent paired comparison. I, schematic illustration of TRPC5–caveolin-1–eNOS complex remodeling upon intracellular Ca 2+ elevation. For B and F , individual data points indicate individual cells examined, and for all the gel images, individual data points indicate technical replicates. Data points are mean ± SD.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Expressing, Clinical Proteomics, Membrane, Comparison, Immunoprecipitation, Construct, Plasmid Preparation, Pull Down Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Plasma membrane dynamics of TRPC5 and eNOS during receptor stimulation. A , representative TIRF images from HEK293 cells expressing eNOS-GFP alone, eNOS-GFP plus TRPC5, or TRPC5-GFP alone upon treatment with 100 μM ATP in 2 mM Ca 2+ -containing solution. B , averaged time courses of surface fluorescence changes (F/F 0 ) obtained by TIRF images from HEK293 cells in each condition upon treatment with 100 μM ATP in 2 mM Ca 2+ -containing solution ( n = 3). C , maximal surface fluorescence changes (ΔF/F 0 ) at 36 to 54 s or 60 to 78 s ( n = 3). p values are determined by one-way ANOVA with subsequent paired comparison. D , representative TIRF images from HEK293 cells expressing eNOS-GFP alone, eNOS-GFP plus TRPC5, or TRPC5-GFP alone upon treatment with 100 μM ATP in 0.5 mM EGTA-containing solution. E , averaged time courses of surface fluorescence changes obtained by TIRF images from HEK293 cells in each condition upon treatment with 100 μM ATP in 0.5 mM EGTA-containing solution ( n = 3–4). F , maximal surface fluorescence changes at 36 to 54 s or 60 to 78 s in 0.5 mM EGTA-containing solution ( n = 3–4). p values are determined by one-way ANOVA with subsequent paired comparison. Data points are mean ± SD. For C and F , individual data points indicate individual cells examined.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Clinical Proteomics, Membrane, Expressing, Fluorescence, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: Selective TRPC5 inhibition suppresses acetylcholine-induced relaxation of rat aorta. A, localization of TRPC5 and eNOS in the endothelium of mesenteric arteries isolated from rat in situ under whole-mount staining visualized by confocal microscopy shown en face. The bar indicates 20 μm. B, localization of TRPC5 and eNOS, and caveolin-1 in the endothelium of mesenteric arteries isolated from rat in situ under whole-mount staining visualized by confocal microscopy. Anti-TRPC5 antibody ( green ), anti-eNOS antibody ( red ), and anti-caveolin-1 antibody ( red ) are used. The nuclei are stained with Hoechst 33342 ( blue ). The bar indicates 20 μm. C , representative time course of acetylcholine-induced relaxation of isolated rat aorta. D , representative time course of acetylcholine-induced relaxation of isolated rat aorta pretreated with 50 μM AC1903. In C and D , 0.3 mM phenylephrine is first added to the bath solutions to induce maximum contraction. E , averaged relaxation of rat aorta (n = 7). Individual data points indicate biological replicates. Data points are mean ± SD.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Inhibition, Isolation, In Situ, Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Dynamic remodeling of TRPC5 channel–caveolin-1–eNOS protein assembly potentiates the positive feedback interaction between Ca 2+ and NO signals

doi: 10.1016/j.jbc.2024.107705

Figure Lengend Snippet: A proposed model for dynamic interaction among TRPC5 channel, caveolin-1, and eNOS coordinating interplay between Ca 2+ and NO signals. A , in the resting state, caveolin-1 provides a scaffold for the assembly of TRPC5 and eNOS into a protein complex. B , upon stimulation of vasodilator receptors, Ca 2+ influx via TRPC5 is induced through the PLC cascade to evoke the primary rise of [Ca 2+ ] i , activating CaM. C , primary increase of [Ca 2+ ] i triggers the plasma membrane translocation of TRPC5 and thus increases the number of TRPC5–caveolin-1–eNOS assembly. D , the assembly is likely to increase the efficiency of the association of eNOS with Ca 2+ -CaM, which releases eNOS from caveolin-1 and allows eNOS to interact with the C-terminus of TRPC5 (residues 936–975). This proposed model enables eNOS to produce NO in the vicinity of TRPC5, optimizing secondary activation of TRPC5 through S -nitrosylation and consequently amplifying Ca 2+ signaling.

Article Snippet: The cells were then incubated for 1 h at room temperature with anti-TRPC5 goat polyclonal antibody (Santa Cruz Biotech C17) and anti-eNOS mouse monoclonal antibody (CALBIOCHEM) or anti-caveolin-1 rabbit polyclonal antibody (Santa Cruz Biotech), each diluted in Can Get Signal immunostain (TOYOBO).

Techniques: Clinical Proteomics, Membrane, Translocation Assay, Activation Assay