Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4

Abstract

Enterochromaffin (_EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuli_EC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details of_EC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouse_EC cells express the mechanosensitive ion channel Piezo2. The mechanosensitive currents in a human_EC cell model QGP-1 were blocked by the mechanosensitive channel blocker D-GsMTx4. In the present study we aimed to characterize the effects of the mechanosensitive ion channel inhibitor spider peptide D-GsMTx4 on the mechanically stimulated currents from both QGP-1 and human Piezo2 transfected HEK-293 cells. We found co-localization of 5-HT and Piezo2 in QGP-1 cells by immunohistochemistry. QGP-1 mechanosensitive currents had biophysical properties similar to dose-dependently Piezo2 and were inhibited by D-GsMTx4. In response to direct displacement of cell membranes, human Piezo2 transiently expressed in HEK-293 cells produced robust rapidly activating and inactivating inward currents. D-GsMTx4 reversibly and dose-dependently inhibited both the potency and efficacy of Piezo2 currents in response to mechanical force. Our data demonstrate an effective inhibition of Piezo2 mechanosensitive currents by the spider peptide D-GsMTx4.

Keywords: D-GsMTx4; Piezo2; enterochromaffin cell; mechanosensitive ion channel; mechanotransduction.

Figure 1.

QGP-1 cells contain 5-HT and express Piezo2. Top row, epifluorescence images of Piezo2 and 5-HT immunohistochemistry with DAPI overlap. Middle row, omission of Piezo2 primary antibody eliminates Piezo2 labeling. Bottom row, omission of 5-HT antibody eliminates 5-HT labeling. Scale bar 50 µm.

Figure 2.

QGP-1 mechanosensitive currents are inhibited by the tarantula peptide D-GsMTx4 in a dose-dependent manner. QGP-1 cells were voltage-clamped and force was applied via graded membrane displacement by a piezo transducer driven glass probe. (A) Typical fast inward QGP-1 mechanosensitive currents in response to 3.5 µm membrane displacement in absence (control, black) and presence of 5 μM (red) and 10 μM (purple) D-GsMTx4. (B) Compared to the peak current in control, there was a significant decrease in peak current when Piezo2 was inhibited by 5 µM D-GsMTx4 (80.2 ± 18.4% inhibition, n = 4, *P < 0.05 by unpaired t-test with Welch's correction).

Figure 3.

Human Piezo2 transiently transfected in HEK-293 cells produce mechanosensitive currents that are inhibited by D-GsMTx4. (A) A stepwise (0.3 µm step) increase in cell membrane deformation resulted in a set of fast activating and inactivating mechanically-induced inward currents, with blue traces highlighting current in response to a maximum stimulus, in both absence (Control) and presence (D-GsMTx4) of 5 µM D-GsMTx4. (B) Peak current-displacement relationship (n = 4) fitted by a two-state Boltzmann function in Control solution (Black, midpoint of 3.5 ± 0.1 µm) and perfused with 5 µM D-GsMTx4 (Red, midpoint of 4.2 ± 0.1 µm). (C) Inward currents elicited by 3.5 µm displacement in control solution (black) are inhibited by 55.7 ± 21.4% after application of 5 µM D-GsMTx4 (red) and washout (Green) restores peak current after exposure with 5 µM D-GsMTx4. (D) Typical inward currents evoked by a 5.0 µm displacement (I_max) in control solution (Black) and application of 5 µM D-GsMTx4 (Red) or 10 µM D-GsMTx4 (purple). A typical non-transfected HEK-293 cell response to mechanical stimulus is shown in gray. (E) Current-concentration whisker plot in presence of 0 (Black, −634.9 ± 53.0 pA), 5 µM (Red, −232.3 ± 37.2 pA) and 10 µM (Blue, −67.6 ± 0.9 pA) D-GsMTx4 (*P < 0.05 by unpaired t-test with Welch's correction).