Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: Treatment of cells with cholesterol increases, but with lovastatin decreases, TRPM4 channel activity by regulating its sensitivity to Ca 2+ in mpkCCD c14 Cells. (A) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 30 μg/ml cholesterol for 48 hrs. From top to bottom: 1 μM, 10 μM, 200 μM, 1 mM, and 5 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (B) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing different concentrations of free Ca 2+ . The cells were treated with 5 μM lovastatin for 48 hrs. From top to bottom: 10 μM, 200 μM, 1 mM, 5 mM and 10 mM free bath Ca 2+ . “C” indicates channel at the closed state; “O” indicates single-level openings. (C) The effect of membrane cholesterol on Ca 2+ -dependence of channel opening. Channel P O was plotted as a function of free Ca 2+ concentration in the bath. P o values are shown for patches either with exogenous cholesterol treatment (black line) or lovastatin treatment (red line). n = 4–7 cells for different data points.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Activity Assay, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: Application of MβCD to the cytoplasmic bath decreases TRPM4 channel activity which is reversed by exogenous cholesterol. (A) A representative single channel recording from an inside-out patch shows TRPM4 activity before and after replacement of control cytoplasmic bath solution first with a solution containing 0.5 mM MβCD and then with a solution containing 30 μg/ml cholesterol. “C” indicates channel at the closed state. “O” indicates single-level openings. “I, II, and III” are zoom-ins of the single-channel recording. (B) Summary plots of TRPM4 channel P O under each indicted condition. n = 5 paired experiments, ** P < 0.01, significantly different with control; ## P < 0.01, significantly different with cells treated with 0.5 mM MβCD.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: Cholesterol stimulates TRPM4 via a PI(4, 5)P 2 -dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCD c14 cells with 20 nM wortmannin had no effect on cholesterol-induced TRPM4 channel activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 6 paired experiments, ** P < 0.01, significantly different with cells treated with 20 nm wortmannin. (C) A representative single channel recording shows that depletion of PI(4, 5)P 2 with 20 μM wortmannin abrogated cholesterol-induced TRPM4 channel activity. (D) Summary plots of TRPM4 channel P O under indicated conditions. n = 5 paired experiments. (E) A representative single channel recording shows that application of 20 μM diC8-PI(4,5)P 2 induced TRPM4 channel activity after cholesterol failed to stimulate TRPM4 in the presence of 2 μM PGE2. (F) Summary plots of TRPM4 channel P O under indicated conditions. n = 4 paired experiments. ** P < 0.01, significantly different with cells treated with 2 μM PGE2 or 30 μg/ml cholesterol.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: PI(4, 5)P 2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P 2 significantly increased TRPM4 activity. (B) Summary plots of TRPM4 channel P O under each indicated conditions. n = 5 paired experiments. ** P < 0.01, significantly different with control. (C) TRPM4 was detected in dots where most anionic phospholipids including PI(4, 5)P 2 are located on PIP Strips membrane. Data represent three individual experiments showing consistent results.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: TRPM4 channels are mainly located in lipid rafts. (A) Representative confocal microscopy images indicate that majority of TRPM4 (green) was co-localized with cholera toxin B (red) in the apical membrane; white rectangular box indicate zoomed-in areas shown in the Zoom-in panels. Data represent five individual experiments showing consistent results (B) Majority of TRPM4 was detected in low-density regions in sucrose gradient experiments; Caveolin-1 was used as a control protein that is known to be located in lipid rafts. Data represent three individual experiments showing consistent results.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCD c14 Cells

doi: 10.3389/fphar.2021.627875

Figure Lengend Snippet: Treatment with cholesterol or lovastatin does not alter expression levels of TRPM4 in mpkCCDc14 Cells. (A) Representative confocal microscopy images of mpkCCDc14 cells stained with TRPM4 antibody under each indicated conditions. (B) Summary plots of fluorescence intensity of TRPM4. Data are from 24 cells in four sets of separate experiments. (C) Representative Western blots from cell-surface biotinylated and the total proteins of TRPM4 protein. (D) Summary plots of relative expression of TRPM4. Cells were either under control conditions or treated with 30 μg/ml cholesterol alone, 30 μg/ml cholesterol plus 5 μM lovastatin, or 5 μM lovastatin alone for 48 hrs, respectively. n = 5. (E) Representative single channel recording from inside-out patches exposed the patch membrane to the bath containing 5 mM CaCl 2 , followed by a bath solution with 10 mM EGTA and no calcium. Cells were either under control conditions or treated with 30 μg/ml cholesterol, or 5 μM lovastatin for 48 hrs, respectively. (F) Summary plots of the number of active channels in the patches under each indicated condition. n = 5 for control cells, n = 5 for cells treated with cholesterol, n = 4 for cells treated with lovastatin.

Article Snippet: Briefly, after fixation with 4% paraformaldehyde at room temperature for 10 min, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% BSA/PBS-T for 30 min. Rabbit polyclonal anti-TRPM4 antibody (alomone ACC-044; 1:100 dilution) was added in 1% BSA/TBS-T for overnight at 4°C.

Techniques: Expressing, Confocal Microscopy, Staining, Fluorescence, Western Blot