Fig 2: Immunofluorescence imaging of TRPM4 in the healthy rat heart. (A) Adult rat heart sections were labeled with anti-TRPM4 antibodies (green) and stained with Hoechst 33342 for the nuclei (blue). The green and blue fluorescence images were overlaid with the DIC image. Staining specificity was confirmed by the lack of signal when anti-TRPM4 antibodies were preincubated with the antigenic blocking peptide (ab65597). Scale bar: 100 µm. (B) Double immunofluorescence showing TRPM4 (green) and SERCA2 (red) expression in ventricular cardiomyocytes. Scale bar: 20 µm. The overlaid image (left bottom) reveals alternating positions for the TRPM4 and SERCA2 proteins on the longitudinal axis of cardiomyocytes. Arrowheads indicate the intercalated disc. Fluorescence intensity profile (right bottom) confirmed the alternating expression of TRPM4 and SERCA2.

Fig 3: Cholesterol stimulates TRPM4 via a PI(4, 5)P2-dependent mechanism. (A) A representative single channel recording shows that treatment of mpkCCDc14 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)P2 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)P2 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.

Fig 4: PI(4, 5)P2 stimulates TRPM4 via a physical interaction. (A) A representative single channel recording shows that diC8-PI(4,5)P2 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)P2 are located on PIP Strips membrane. Data represent three individual experiments showing consistent results.

Fig 5: Neuronal expression of TRPM4 in mouse prefrontal cortex (PFC). (A) Panoramic view of the frontal cortex showing double immunofluorescence labeling for TRPM4 (green) and NeuN (red) in a coronal mouse brain section at P35. The inset shows a coronal map of this area. (B) Confocal images of layers 1 and 2/3 of mPFC showing the expression of TRPM4 (top), NeuN (middle), and the merge of both signals (bottom) showing the expression in the soma and neurites (arrows). Scale bar: A = 300 µm; B = 40 µm. (C) Representative I/V ramps (–80 to 80 mV, 0.16 mV/ms) recorded in the soma of pyramidal neurons from layer 2/3 at mPFC. Black traces represent the basal current, red traces represent the effect of 10 µM 9-Ph (whole bath perfusion), and blue traces represent intracellular EGTA (10 mM). The right panel shows the quantification of the current at –80 mV (arrow) (n = 8). All experiments were performed in the presence of glutamatergic (CNQX, AP5) and GABAergic blockers (Picrotoxin).