Piezo1 Channels Are Inherently Mechanosensitive

Abstract

The conversion of mechanical force to chemical signals is critical for many biological processes, including the senses of touch, pain, and hearing. Mechanosensitive ion channels play a key role in sensing the mechanical stimuli experienced by various cell types and are present in organisms from bacteria to mammals. Bacterial mechanosensitive channels are characterized thoroughly, but less is known about their counterparts in vertebrates. Piezos have been recently established as ion channels required for mechanotransduction in disparate cell types in vitro and in vivo. Overexpression of Piezos in heterologous cells gives rise to large mechanically activated currents; however, it is unclear whether Piezos are inherently mechanosensitive or rely on alternate cellular components to sense mechanical stimuli. Here, we show that mechanical perturbations of the lipid bilayer alone are sufficient to activate Piezo channels, illustrating their innate ability as molecular force transducers.

Keywords: Piezo1; lipid bilayer; mechanosensitive ion channel; mechanotransduction; membrane asymmetry; membrane tension.

Figure 1. Reconstitution of Piezo1 in droplet lipid-bilayers

(A) Purified Piezo1-FLAG separated on Bis-Tris native gel and visualized by Coomassie staining (representative gel from n=5). (B–D) Single-channel current recordings in 500 mM KCl, V= −100 mV and the all-point histograms of purified Piezo1-WT (B–C) and Piezo1-E2133A mutant (D). Channel openings are downward deflections where c=closed and o=open. The insets below show the channel block upon RR injection (40 μM final). (E) Single-channel conductance comparison of Piezo1-WT vs Piezo1-E2133A in LPA||PC lipid-bilayers under similar conditions (500 mM KCl, 10 mM HEPES, pH 7.4 at V= −100 mV). The two groups in question are significantly different as determined by two-tail unpaired t-test: P value < 0.001. Error bars represent SEM. See also Table S1.

Figure 2. Activation of purified Piezo1 by osmotic stress stimulation

(A–D) Experimental illustrations of droplets with and without an osmotic gradient either in the presence of 500 mM KCl or 70 mM KCl and the single-channel recordings of Piezo1 in PC||PC bilayers at V= −100 mV. (E) Single-channel conductance comparison of Piezo1 for the indicated experimental conditions. The two groups in question are significantly different as determined by two-tail unpaired t-test: P value < 0.001. Error bars represent SEM. (F–G) Single-channel recordings of MscS in PC||PC bilayers in the absence and presence of osmotic stress with 200 mM KCl at V= +30 mV. (H–I) Single-channel recordings of KcsA in the indicated pH solutions in the absence and presence of osmotic stress with 200 mM KCl at V= +100 mV. See also Table S1 and Figure S1.

Figure 3. Activation of Piezo1 by solvent injection in the lipid monolayer

(A–B) Illustration of the solvent injection assay and electrical recordings in the absence of protein before and after the injection-protocols. (C–I) Illustration of the solvent injection assay and examples of electrical recordings in the presence of (C–E) Piezo1 in 500 mM KCl at V=−100mV, (F–G) MscS in 200 mM KCl at V= +30 mV and (H–I) KcsA in 200 mM KCl, V=+100mV. See also Table S1, Figure S2 and Supplemental Experimental Procedures.