TMEM150C/Tentonin3 Is a Regulator of Mechano-gated Ion Channels

Abstract

Neuronal mechano-sensitivity relies on mechano-gated ion channels, but pathways regulating their activity remain poorly understood. TMEM150C was proposed to mediate mechano-activated current in proprioceptive neurons. Here, we studied functional interaction of TMEM150C with mechano-gated ion channels from different classes (Piezo2, Piezo1, and the potassium channel TREK-1) using two independent methods of mechanical stimulation. We found that TMEM150C significantly prolongs the duration of the mechano-current produced by all three channels, decreases apparent activation threshold in Piezo2, and induces persistent current in Piezo1. We also show that TMEM150C is co-expressed with Piezo2 in trigeminal neurons, expanding its role beyond proprioceptors. Finally, we cloned TMEM150C from the trigeminal neurons of the tactile-foraging domestic duck and showed that it functions similarly to the mouse ortholog, demonstrating evolutionary conservation among vertebrates. Our studies reveal TMEM150C as a general regulator of mechano-gated ion channels from different classes.

Keywords: K2P2.1; Piezo1; Piezo2; TMEM150C; TREK-1; Tentonin3; mechano-receptor; mechano-sensitivity; mechano-transduction; trigeminal ganglia.

Figure 1. Mouse and Duck TMEM150C Potentiate Piezo2 Mechano-current in HEK293T^ΔP1 Cells

(A and B) Representative images of RNA in situ hybridization (A) and quantification of TMEM150C-expressing neurons (B) in adult duck TG (2,808 cells from 19 TG sections from 2 animals). Scale bar, 50 µm. (C) Exemplar whole-cell MA current traces recorded in HEK293T^ΔP1 cells expressing Piezo2 with or without mouse or duck TMEM150C in response to mechanical indentation with a glass probe to the indicated depth (E_hold = −80 mV). (D) Peak MA current measured at different indentation depths in HEK293T^ΔP1 cells expressing indicated constructs (E_hold = −80 mV). Data shown as mean ± SEM. (E) Quantification of MA current activation threshold (p < 0.0001, one-way ANOVA with Dunnett’s correction, **p < 0.001, ****p < 0.0001). Data shown as mean ± SEM. (F) Quantification of MA current inactivation rate (τ_inact) measured at different indentation depths (ordinary two-way ANOVA with Bonferroni correction, p < 0.0001 for expression construct effect; NS, not significant; p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001; red, blue, and gray asterisks indicate statistical comparisons between, respectively, Piezo2/mTMEM150C and Piezo2, Piezo2/dTMEM150C and Piezo2, and Piezo2/mTMEM150C and Piezo2/dTMEM150C). Data shown as mean ± SEM. (G) Representative traces of whole-cell MA currents evoked in response to 9 µm mechanical indentation at different voltages from −100 mV to 100 mV, in 20 mV increments. (H) Quantification of MA current τ_inact at different voltages (two-way ANOVA with Sidak’s correction, *p < 0.05, **p < 0.01, ****p < 0.0001). Data shown as mean ± SEM. (I) Peak MA current-voltage plots in response to mechanical indentation of 5–10 µm for Piezo2/GFP and 4-9 µm for Piezo2/mTMEM150C. The inset shows quantification of the reversal potential E_rev (unpaired t test; NS, not significant; p > 0.05). Data shown as mean ± SEM. See also Figures S1, S3A, and S3B.

Figure 2. TMEM150C Potentiates MA Current Produced by Piezo1

(A) Exemplar whole-cell MA current traces recorded in HEK293T^ΔP1 cells in response to mechanical indentation with a glass probe at E_hold = −80 mV. Arrow indicates the position of persistent post-stimulus MA current measurement. (B) Peak MA current measured at different indentation depths in HEK293T^ΔP1 cells expressing indicated constructs (E_hold = −80 mV). Data shown as mean ± SEM. (C and D) Quantification of MA current activation threshold (C) and inactivation rate τ_inact (D). NS, not significant; p > 0.05, **p < 0.01; unpaired t test (C) and Mann-Whitney U-test (D). Data shown as mean ± SEM. (E) Post-stimulus MA current amplitude at different indentation depths. ****p < 0.0001 for expression construct effect, two-way ANOVA. Data shown as mean ± SEM. (F) Quantification of peak-normalized amplitude of persistent post-stimulus MA current. ****p < 0.0001, Mann-Whitney U test. Data shown as mean ± SEM. See also Figure S2A.

Figure 3. TMEM150C Potentiates Piezo1 MA Current Evoked by High-Speed Pressure Clamp

(A) Exemplar cell-attached MA current traces recorded in HEK293T^ΔP1 cells in response to application of a negative pressure in the pipette at E_hold = −60 mV. Each pressure step was preceded by a 500-ms step at 5 mmHg to remove inactivation. Arrow indicates the position of persistent post-stimulus MA current measurement. (B) Quantification of peak MA current amplitude measured at −60 mV at different pressures. Data shown as mean ± SEM. (C and D) Quantification of MA current activation threshold (C) and inactivation rate τ_inact (D). NS, not significant; p > 0.05, **p < 0.01; Mann-Whitney U-test (C) and unpaired t test (D). Data shown as mean ± SEM. (E) Post-stimulus MA current amplitude at different pressures. ****p < 0.0001 for expression construct effect, two-way ANOVA. Data shown as mean ± SEM. (F) Quantification of peak-normalized amplitude of persistent post-stimulus MA current. *p < 0.05, Welch’s test. Data shown as mean ± SEM. See also Figure S2B.

Figure 4. TMEM150C Potentiates MA Current of Potassium-Selective TREK-1 Channel

(A) Exemplar cell-attached MA current traces recorded in HEK293T^ΔP1 cells in response to application of a negative pressure in the pipette using a high-speed pressure clamp (E_hold = 0 mV). Each pressure step was preceded by a 500-ms step at 5 mmHg to remove inactivation. (B) Quantification of peak MA potassium current amplitude measured at E_hold = 0 mV at different pressures. Data shown as mean ± SEM. (C and D) Quantification of cell-attached MA current activation threshold (C) and average inactivation rate (D). NS, not significant; p > 0.05, **p < 0.01, Mann-Whitney U test. Data shown as mean ± SEM. See also Figures S2C and S3C.