Figure 4. Mutations of a conserved Glutamate residue alter mPiezo1 pore properties.

(a) Protein sequence surrounding E2133 with putative topology models of mPiezo1 C-ter region. E2133 is highlighted (orange) and green bars indicate hydrophobic regions. (b) Representative (from 4–7 experimental replicates) stretch-activated channel openings at −80 mV from cells transfected with mPiezo1 WT, E2133Q, E2133D and E2133K. Stimulation intensities are −15, −10, −20 and −50 mm Hg, respectively. (c) Average I-V relationships of stretch-activated single channels in cells transfected with mPiezo1 WT, E2133Q, E2133D and E2133K (n = 7, 5, 5 and 4, respectively; mean ± s.e.m.). Single-channel amplitude was determined as the amplitude difference in Gaussian fits of full-trace histograms. (d) Unitary conductance calculated from the slope of linear regression line of individual cells (mean ± s.e.m.; One-way ANOVA with Dunn’s comparison to WT, **P<0.01). (e) Representative (from 6–9 experimental replicates) traces of MA currents recorded with 150 mM CsCl based intracellular solution and 100 mM CaCl₂ extracellular solution from mPiezo1 WT (black), E2133Q (grey), E2133D (red) and E2133K (blue) transfected cells. Currents are elicited from -69.6 to +50.4 mV, Δ20 mV. Scale bars: 100 pA, 50 ms. Probe stimulation displacements are 3, 4, 6 and 7 μm, respectively. (f) Average I-V relationships of MA currents recorded from mPiezo1 WT, E2133Q, E2133D and E2133K expressing cells with 150 mM CsCl based intracellular solution and 100 mM CaCl₂ extracellular solution (n = 8, 6, 6 and 9, respectively). Inset: Average reversal potentials from individual cells corresponding to panel (f) experiments (WT: 10.3 ± 0.6 mV; E2133Q: 5.0 ± 0.5 mV; E2133D: 12.1 ± 1.0 mV and E2133K: 2.6 ± 0.4 mV; mean ± s.e.m.; One-way ANOVA with Dunn’s comparison to WT, **P<0.01). Panels (b), (c) and (d) experiments were performed in cell-attached configuration with Na⁺ as the only permeating cation in the recording pipette.