Journal: Circulation Research

Article Title: Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

doi: 10.1161/circresaha.115.303831

Figure Lengend Snippet: Figure 5. Transient receptor potential canonical (TRPC) channels colocalize with l-type Ca2+ channel (LTCC) in caveolae membrane microdomains where their organization is required for hypertrophic signaling. A, Plasma membranes (PMs) were purified from total cell homogenates (H) of isolated myocytes from dominant-negative TRPC4 mice. Immunoprecipitations (IPs) and Westerns were performed with the indicated antibodies (B indicates bound fraction; and U, unbound fraction). B, Sucrose density gradient fractionation on purified PMs from isolated adult feline myocytes (AFMs) infected with Ad-TRPC3 confirms the presence of LTCC and TRPC3 channels in caveolin-3 (Cav3)–enriched lipid raft membrane fractions along with the hypertrophic effector calcineurin (Cn; fraction 1 to fraction 11, F1–F11). C, AFMs were infected with Ad-NFAT-GFP and the indicated adenoviruses and NFAT translocation was monitored in response to the TRPC agonist OAG (10 umol/L) in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mmol/L). Scale bar is 10 μm. Average data are represented in D as the nuclear to cytoplasmic GFP ratio of n=100 cells per condition. P<0.05 was considered significant with *P≤0.05; **P≤0.001; ***P≤0.001 vs red fluorescent protein control; ##P≤0.001; ###P≤0.001 vs TRPC3; &&P≤0.001; &&&P≤0.001 vs TRPC6.

Article Snippet: Sheep anti-rabbit IgG-coated magnetic beads (Dynal Biotech) were pre-incubated with an antibody for TRPC4 (Alomone Labs), TRPC6 (Alomone Labs) or Cav1.2 (LTCC α1C, Millipore) for 2-4 hours at room temperature.

Techniques: Membrane, Clinical Proteomics, Purification, Isolation, Dominant Negative Mutation, Fractionation, Infection, Translocation Assay, Control

Journal: Biochimica et biophysica acta

Article Title: Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

doi: 10.1016/j.bbamcr.2015.07.011

Figure Lengend Snippet: Fig. 1. Sdc4 modulates podocyte TRPC6 channels. (a) Knockdown of endogenous Sdc4 using siRNA reduces steady-state surface expression of TRPC6 in immortalized mouse podocytes. Blots on the left are results of representative cell-surface biotinylation assay. Note substantial reduction of surface TRPC6, and total Sdc4 in cells transfected with siRNA targeting Sdc4 com- pared to cells transfected with non-targeting siRNA. Bar graphs to the right show densitometric analyses of three repetitions of this experiment. In this and subsequent figures, bars rep- resent mean ± standard deviation except as noted below. Ordinates represent fold changes in signal compared to control.(b) Overexpression of Sdc4 increases steady-state surface expression of TRPC6. Representative cell-surface biotinylation assay and densitometric analysis is shown below the blot. (c) Exposing podocytes to recombinant Sdc4 ectodomain (20 ng/ml) for 24 h caused a marked increase in steady-state surface expression of TRPC6. (d) Representative whole-cell recordings from podocytes showing cationic currents evoked by 100 μM OAG and blockade of these currents by 50 μM La3+. Traces shown are of cationic currents recorded during application of voltage ramps (−80 mV to +80 mV over 2.5 s). Note that cationic current in the presence of OAG is larger in the Sdc4-treated cell. (e) Summary of the fold increase in current at +80 mV evoked by OAG over baseline in control cells and cells exposed to Sdc4 ectodomain for 24 h. This bar graph plots mean ± s.e.m. from 10 cells in each group. Asterisk indicates P b 0.05 by Student's unpaired t-test.

Article Snippet: Proteins in all of these fractions were then quantified by immunoblot as described above using a polyclonal antibody against TRPC6 purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Knockdown, Expressing, Cell Surface Biotinylation Assay, Transfection, Standard Deviation, Control, Over Expression, Recombinant

Journal: Biochimica et biophysica acta

Article Title: Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

doi: 10.1016/j.bbamcr.2015.07.011

Figure Lengend Snippet: Fig. 2. Sdc4 interacts with β3-integrin in podocytes. (a) Representative experiment showing that β3-integrin can be detected in the material precipitated by an antibody against Sdc4, but not by a control IgG. (b) Confocal immunofluorescence showing co-localization of Sdc4 and β3-integrin in immortalized podocytes. These cells were not permeabilized before exposure to primary antibodies. (c) Representative immunoblot showing that 24 h exposure to Sdc4 ectodomain increases total expression of β3-integrin in podocytes (left) and densitometric analysis of three repetitions of this experiment (right). (d) In contrast to β3-integrin, TRPC6 channels are not detected in the material precipitated by an antibody against Sdc4.

Article Snippet: Proteins in all of these fractions were then quantified by immunoblot as described above using a polyclonal antibody against TRPC6 purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Control, Western Blot, Expressing

Journal: Biochimica et biophysica acta

Article Title: Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

doi: 10.1016/j.bbamcr.2015.07.011

Figure Lengend Snippet: Fig. 4. Role of integrins on surface expression of TRPC6 channels evoked by Sdc4 ectodomain. Effects of Sdc4 ectodomain on steady-state surface expression of podocyte TRPC6 (a) and total expression of β3-integrin (b) are blocked by treatment with 1 μM cilengitide (CGT), an inhibitor of outside-in signaling through integrins that contain αv subunits, such as αvβ3-integrin.

Article Snippet: Proteins in all of these fractions were then quantified by immunoblot as described above using a polyclonal antibody against TRPC6 purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing

Journal: Biochimica et biophysica acta

Article Title: Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

doi: 10.1016/j.bbamcr.2015.07.011

Figure Lengend Snippet: Fig. 6. Signaling pathways in the mobilization of TRPC6 channels evoked by Sdc4. (a) Effects of Sdc4 on steady-state surface expression of TRPC6 are blocked by TEMPOL, a membrane permeable ROS quencher and superoxide dismutase mimetic agent. Effects of Sdc4 on the trafficking of TRPC6 persist in the presence of 20 μM cyclosporine A, an inhibitor of calcineurin (b) or the NFATc1 inhibitor 11R-VIVIT (10 μM) (c). (d) The effects of Sdc4 ectodomain on surface expression of TRPC6 were blocked by 50 μM NS-23766, an inhibitor of Rac1-mediated signaling. (e) Application of 1 μM membrane-permeable C3 transferase (C3T), an inhibitor of Rho enzymes, caused an increase in steady state surface expression of TRPC6 but did not exert inhibitory or additive effects with Sdc4 ectodomain.

Article Snippet: Proteins in all of these fractions were then quantified by immunoblot as described above using a polyclonal antibody against TRPC6 purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Protein-Protein interactions, Expressing, Membrane

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Design and generation of XLone transgenic HeLa cells. a Schematic design showing the transposable plasmid cassette ( 5′ PB 5′ PiggyBac Terminal Repeat; 3′ PB 3′ PiggyBac Terminal Repeat; MCS multiple cloning sites; Bsd blasticidin resistance gene; EFS EF1 alpha promoter; TRE3G tetracycline responsive element 3G promoter). b Flow cytometry analysis of the cells after Bsd drug selection and FACS-live cell sorting of respective wild type, P112Q, G757D, and V691Kfs*. c Representative images illustrate the expression of YFP reporter (green) after transfection, Bsd selection, and FACs sorting, as shown by the merged image of the bright field and YFP signal for each of the variants studied. Scale bar 50 µm. d Representative FACS plots of transgenic HeLa cells demonstrate an enriched population up to a maximum of 89% YFP-positive cells. e Western blot for TRPC6 protein from wild-type, p112Q, G757D, and V691kfs* (~ 130 kDa) (B) β-actin (~ 45 kDa) in HeLa cells

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Transgenic Assay, Plasmid Preparation, Clone Assay, Flow Cytometry, Selection, FACS, Expressing, Transfection, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Tridimensional cryo-EM structure of tetrameric TRPC6 (PDB: 6UZ8) indicating novel truncated p.Val691Lysfs*2 (V691Kfs*) ([STOP]AA692) protein in exon 8. a View from the extracellular side, representing four monomeric TRPC6 subunits forming the ion channel complex. b Close-up view of the helices shows an exchange of Valine to Lysine at amino acid position p.691 and in a stop codon at p.692 making a truncated protein. c Full homology view parallel to the membrane. The location of the V691 protein is indicated in dark blue color and red color illustrates the truncated part of the closed channel. d Truncation results in complete loss of the C-terminal helices. e Verification of c.2060_2070dup AAGTGAAATCA variant compared with wild-type TRPC6 sequence. f Comparison of variant sequence with nucleotide insertion and addition of stop codon at p.692, with wild-type TRPC6 sequence

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Cryo-EM Sample Prep, Variant Assay, Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Functional characterization reveals GOF, LOF, and truncated phenotype in FSGS-related TRPC6 mutations. Changes in intracellular Ca 2+ concentration ( F / F 0 ) were measured in Fluo-4 AM-loaded HeLa cells pretreated with doxycycline (Dox) (24 h prior) expressing either wild type (WT), P112Q, G757D, or V691Kfs*. Each condition was carried out in replicates, while un-transfected HeLa cells served as control. a Carbachol (Cch) (100 µM) was added 20 s after the start of the measurement. Representative measurement for all samples is shown in the upper panel, while a summary is presented as a bar graph ( n = 3, mean ± SEM) in the lower panel. The graph shows the differences in amplitudes of the responses to Cch. b DOG (100 µM) was added 20 s after the start of the measurement. Representative measurement for all samples is shown in the upper panel, while a summary is presented as a bar graph ( n = 3, mean ± SEM) in the lower panel. The graph shows the differences in amplitudes of the responses to DOG

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Functional Assay, Concentration Assay, Expressing, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Functional characterization of disease-related TRPC6 mutants in calcium influx measurements. Changes in intracellular calcium concentration ( F / F 0 ) were measured in Fluo-4-AM-loaded HeLa cells expressing wild type (WT), P112Q, G757D, or V691Kfs* treated with doxycycline (Dox) (0.5 µg/mL) 24 h before measurement and no Dox treated cells. a – d Treatment with agonist carbachol (Cch: 100 µM): Cch was added about 20 s after the start of the measurement. Effects of TRPC6 blockers SAR7334 (SAR; 100 µM) and SH045 (100 µM) are observed in each TRPC6 mutant. e – h DOG (100 µM) was added about 20 s after the start of the measurement. Effects of SAR7334 (SAR; 100 nM) and SH045 (100 nM) are observed in each TRPC6 mutant

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Functional Assay, Concentration Assay, Expressing, Mutagenesis

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Electrophysiologic characterization revealed currents mediated by WT TRPC6, TRPC6 V691Kfs*, TRPC6 P112Q, and TRPC6 WT + V691Kfs*. Whole-cell currents were recorded in transfected HEK293 cells using the protocol described in the “Materials and methods” section. After obtaining the whole-cell configuration, currents from −100 to + 100 mV were recorded during voltage ramps. a , c , e , h Inward currents at −60 mV were plotted over time. b , d , f , i Current–voltage relationships obtained during voltage ramps from −100 to + 100 mV: 1, basal currents (black); 2, 100 µM carbachol (Cch)-induced currents (red); and 3, 100 µM Cch-induced currents in the presence of 100 µM gadolinium chloride (Gd 3+ , blue). a , b Currents were measured in WT TRPC6 cells. c , d Currents were recorded in TRPC6 V691Kfs*-transfected cells. e , f Currents were recorded in TRPC6 P112Q-expressing cells. h , i Currents were recorded in TRPC6 WT + V691Kfs*-transfected cells. g Statistical analysis of the Cch-activated currents (plotted as the current density at −60 mV). Data are means ± SEMs in each group: six cells (WT TRPC6, black), seven cells (truncated TRPC6), eight cells (TRPC6 WT + V691Kfs*), and five cells (TRPC6 P112Q, gray). * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Transfection, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Co-transfection strategy to characterize the dominant phenotype of TRPC6 mutants. Cells were transfected with mixtures of TRPC6 Wild Type (WT) C-terminally fused to YFP and mCherry, G757D C-terminally fused to YFP and P112Q fused to mCherry (mCh +). a – c Representative flow cytometry plots show the gating strategy to sort double-positive cells expressing both YFP and mCherry (YFP + mCh + , top right) for each co-transfected cells and the remaining 3 gates to sort cells expressing only mChr + (Top left), YFP (bottom right), and non-transfected cells (bottom left). Microscopic images of ( a ′) sorted cells expressing both WT-YFP/P112Q-mCh represented in combined image as YFP + mCh + , ( b ′) sorted cells expressing both G757D-YFP/WT-mCh represented in combined image as YFP + mCh + , ( c ′) sorted cells expressing both G757D-YFP/P112Q-mCh represented in combined image as YFP + mCh + . Scale bar 100 µm

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Cotransfection, Transfection, Flow Cytometry, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis

doi: 10.1007/s00018-023-04901-w

Figure Lengend Snippet: Co-transfection strategy to characterize the dominant phenotype of TRPC6 mutants. Changes in intracellular calcium concentration ( F / F 0 ) were measured in Fluo-4-AM-loaded HeLa cells expressing wild-type (WT), P112Q/WT, G757D/WT, or P112Q/ G757D TRPC6 C-terminally fused to YFP and mCherry treated with doxycycline (Dox) (0.5 µg/mL) 24 h before measurement. a – c Carbachol (Cch: 100 µM) was added about 20 s after the start of the measurement. Effects of SAR7334 (SAR; 100 µM) and SH045 (100 µM) are observed in each TRPC6 mutant. d – f DOG (100 µM) was added about 20 s after the start of the measurement. Effects of SAR7334 (SAR; 100 µM) and SH045 (100 µM) are observed in each TRPC6 mutant. g Statistical analysis of data obtained from at least three independent experiments is presented as bar graphs. The graphs show the differences in amplitudes of the responses on Cch application h Statistical analysis of data obtained from at least three independent experiments is presented as bar graphs. The graphs show the differences in amplitudes of the responses to the DOG application ( n = 3, mean ± SEM). *** P ≤ 0.0001

Article Snippet: The membrane was blocked by Intercept T20 Antibody Diluent (LI-COR Bioscience) for 1 h at room temperature on the shaker and incubated with rabbit anti-TRPC6 (Merck Millipore 1:300) antibody and Rabbit ß-actin (New England Biolabs GmbH, clone 13E5, 1:1000) in Intercept T20 antibody diluent (LI-COR Biosciences) at 4 °C overnight.

Techniques: Cotransfection, Concentration Assay, Expressing, Mutagenesis