Figure 1.
Overexpression of TMEM150C in N2APiezo1−/−cells does not evoke mechanosensitive currents. (A−C, and E) Top: Cartoons of the different mechanical stimuli applied in this study together with representative traces from mechanosensitive currents evoked using indentation (A), HSPC (C), or pillar arrays (E) methods. Piezo and Trpv4 were used as positive controls. Cells overexpressing Tmem150c did not display mechanosensitive currents in any of the assays that differed from cells transfected with the empty expression vector. RE, recording electrode; MS, mechanical stimulator. A, C, and E quantified for all stimulus strengths in B, D, and F. (A and B) Scatter plots showing the maximal peak (Imax) from mechanosensitive currents observed in N2aPiezo1−/− cells transfected with vector, Tmem150c or Piezo2. No mechanosensitive currents were observed for N2APiezo1−/− transfected with Tmem150c (Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001) with indentation (A and B) or HSPC (C and D); Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; Piezo1 vs. vector P = 0.0003; Piezo1 vs. Tmem150c P = 0.0002. (E) N2APiezo1−/− cells transfected with vector controls occasionally displayed a pillar induced mechanosensitive current, but the frequency of such currents was not higher in cells expressing Tmem150c. In contrast, almost all cells overexpressing either Piezo2 or Trpv4 showed large and robust mechanosensitive currents to pillar deflection, Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; vector vs. Piezo2 P = 0.0007; vector vs. Trpv4 P = 0.003; Tmem150c vs. Piezo2 P = 0.002; Tmem150c vs. Trpv4 P = 0.01. (E and F) Current kinetics observed in N2APiezo1−/− overexpressing a vector, Tmem150c or Piezo2 channels. Left: Indentation–current amplitude relationship (A) showing that Tmem150c is indentation-insensitive. Two-way ANOVA, Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001. (C and D) Overexpression of Tmem150c was not associated with the pressure activated currents in membrane patches subjected to pressure pulses. Two-way ANOVA, Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001. Current amplitude–deflection relationship (I) for currents recorded in cells expressing Tmem150c, Piezo2, or Trpv4. (F) Cells expressing Tmem150c exhibited similar amplitude–deflection currents as cells transfected with the empty vector. Two-way ANOVA, Tmem150c vs. vector P > 0.9999; vector vs. Piezo2 P = 0.0003; vector vs. Trpv4 P = 0.0136. *P ≤ 0.05, **P > 0.01, ***P < 0.001, ****P < 0.0001.

Overexpression of TMEM150C in N2A Piezo1−/− cells does not evoke mechanosensitive currents. (A−C, and E) Top: Cartoons of the different mechanical stimuli applied in this study together with representative traces from mechanosensitive currents evoked using indentation (A), HSPC (C), or pillar arrays (E) methods. Piezo and Trpv4 were used as positive controls. Cells overexpressing Tmem150c did not display mechanosensitive currents in any of the assays that differed from cells transfected with the empty expression vector. RE, recording electrode; MS, mechanical stimulator. A, C, and E quantified for all stimulus strengths in B, D, and F. (A and B) Scatter plots showing the maximal peak (Imax) from mechanosensitive currents observed in N2aPiezo1−/− cells transfected with vector, Tmem150c or Piezo2. No mechanosensitive currents were observed for N2APiezo1−/− transfected with Tmem150c (Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001) with indentation (A and B) or HSPC (C and D); Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; Piezo1 vs. vector P = 0.0003; Piezo1 vs. Tmem150c P = 0.0002. (E) N2APiezo1−/− cells transfected with vector controls occasionally displayed a pillar induced mechanosensitive current, but the frequency of such currents was not higher in cells expressing Tmem150c. In contrast, almost all cells overexpressing either Piezo2 or Trpv4 showed large and robust mechanosensitive currents to pillar deflection, Kruskal–Wallis test, Tmem150c vs. vector P > 0.9999; vector vs. Piezo2 P = 0.0007; vector vs. Trpv4 P = 0.003; Tmem150c vs. Piezo2 P = 0.002; Tmem150c vs. Trpv4 P = 0.01. (E and F) Current kinetics observed in N2APiezo1−/− overexpressing a vector, Tmem150c or Piezo2 channels. Left: Indentation–current amplitude relationship (A) showing that Tmem150c is indentation-insensitive. Two-way ANOVA, Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001. (C and D) Overexpression of Tmem150c was not associated with the pressure activated currents in membrane patches subjected to pressure pulses. Two-way ANOVA, Piezo2 vs. vector, P < 0.0001; Piezo2 vs. Tmem150c P < 0.0001. Current amplitude–deflection relationship (I) for currents recorded in cells expressing Tmem150c, Piezo2, or Trpv4. (F) Cells expressing Tmem150c exhibited similar amplitude–deflection currents as cells transfected with the empty vector. Two-way ANOVA, Tmem150c vs. vector P > 0.9999; vector vs. Piezo2 P = 0.0003; vector vs. Trpv4 P = 0.0136. *P ≤ 0.05, **P > 0.01, ***P < 0.001, ****P < 0.0001.

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