A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells

Abstract

TMEM16A is a Ca²⁺-activated Cl^- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Keywords: Ani9; Piezo1; TMEM16A; TMEM16inh-A01; Yoda1; myogenesis; skeletal muscle.

Figure 1

Block of TMEM16A chloride currents by Ani9. (A) Representative currents elicited by 1000 ms test potentials between −100 and +80 mV in the same myocyte before (Ctrl, black) and after the addition of 1 µM Ani9 (+Ani9, gray) and the corresponding Ani9-sensitive currents (magenta) calculated by subtraction. On top, the stimulation protocol used to elicit currents. (B) The I-V relationships in absence and presence of Ani9. (C) The histogram shows the blockage exerted by Ani9 that was significant only for the outward currents at +80 mV (n = 8, ** p = 0.002).

Figure 2

Ani9-sensitive currents are permeable to chloride. (A) Representative I-V relationships of Ani9-sensitive currents recorded in standard (magenta) and low [Cl⁻]_e bath (ochre) solutions. (B) The histogram shows the significant shift of the reversal potential towards a more positive value in low [Cl⁻]_e. (n = 8, ** p = 0.006).

Figure 3

Detection of T16Ainh-A01-sensitive currents. (A) Representative currents recorded in absence (Ctrl, black) and in presence of 5 µM T16Ainh-A01 (+T16inh-A01, blue) and corresponding T16inh-A01-sensitive currents (green) calculated by subtraction. (B) In the presence of the blocker, there is a significant reduction in the currents recorded both at −80 mV and +80 mV (n = 6, * p = 0.03). (C) I-V relationship of T16inh-A01-sensitive currents (n = 5).

Figure 4

The effect of Ani9, a TMEM16A antagonist, on the myocyte fusion index. (A) Representative optical fields where nuclei are stained in 3-day myocyte cultures in control conditions and in the presence of 5 μM Ani9. Nuclei staining is shown as DAPI fluorescence overlay on corresponding bright-field images. Myotubes are indicated by white arrowheads. Scale bar, 50 μm. (B) A reduction in the mean fusion index was observed at the concentrations of 5 μM (**** p < 0.0001) and 10 µM Ani9 (**** p < 0.0001) with respect to control. Ani9 3 μM vs. 5 μM, p < 0.0001; Ani9 3 μM vs. 10 μM, p < 0.0001; Ani9 5 μM vs. 10 μM, p = 0.0005.

Figure 5

The effect of Piezo1 channel activation on TMEM16A. (A) Representative Western blot revealing TMEM16A and Piezo1 presence in myocyte lysate (2 preparations). (B) Representative currents elicited in control (black) and after 2 min in Yoda1 (3 µM, red). (C) Mean I-V curves before and after the addition of Yoda1 (n = 8). (D) Note the significant (** p = 0.008) Yoda1-induced increase in the current amplitude at positive potentials of +80 mV.

Figure 6

Yoda1 effect on TMEM16A currents. (A) I-V relationships of control (Ctrl) in presence of 5 µM T16Ainh-A01 (T16Ainh) or 5 µM Yoda 1 plus 5 µM T16Ainh-A01 (T14Ainh + Yoda1). (B) The TMEM16A blocker T16Ainh-A01 significantly affected the currents recorded at both −80 mV (* p = 0.04) and +80 mV (* p = 0.03). Note that the addition of Yoda1 did not induce significant changes in the presence of T16inh-A01 both at −80 mV (p = 0.74; n = 5) and +80 mV (p = 0.6; n = 5).