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. 2015 May 26;6:7223. doi: 10.1038/ncomms8223

Figure 6. Lysine substitution of E2416 alters pore properties of mPiezo2 channels.

Figure 6

(a) Conservation of mPiezo1 E2133 at position 2,416 on mPiezo2 protein. (b) Representative (from nine and six experimental replicates) stretch-activated channel openings at −80 mV from cells transfected with mPiezo2 WT and E2416K. Stimulation intensities are −15 and −20 mm Hg, respectively. (c) Average I–V relationships of stretch-activated single channels in mPiezo2 WT and E2416K transfected cells (n=9 and 6, respectively; mean±s.e.m.). Single-channel amplitude was determined as the amplitude difference in Gaussian fits of full-trace histograms. (d) Single-channel conductance calculated from the slope of linear regression line of individual cell single-channel I–V relationships (mean±s.e.m.; Mann–Whitney test, ***P<0.001). (e) Average I–V relationships of MA currents recorded from mPiezo2 WT and E2416K expressing cells with 150 mM NaCl-based intracellular solution and 30 mM NaCl extracellular solution. Top left inset: typical recording traces for WT (black) and E2416K (green) from −71.7 to +48.3 mV, Δ20 mV. Scale bars, 100 pA, 50 ms. Probe stimulation displacements are 9 and 8 μm, respectively. Bottom right inset: average reversal potential (mean±s.e.m.; n=6 and 5, respectively; Mann–Whitney test **P<0.01). (f) PCl/PNa permeability ratios of MA currents from cell transfected with mPiezo2 WT, and E2416K (mean±s.e.m.; n=6 and 5, respectively; Mann–Whitney test **P<0.01). (g) Representative (from five experimental replicates) MA current traces from mPiezo2 WT and E2416K transfected cells at −80 mV before, during or after application of 30 μM RR. Each trace is an average of two to four trials. Probe stimulation displacements are 5 and 7 μm, respectively. Scale bars, 100 pA, 100 ms. (h) Average block of MA currents at −80 mV by 30 μM RR in mPiezo2 WT and E2416K transfected cells (n=5 for each; mean±s.e.m.; Mann–Whitney test, **P<0.01). Experiments shown in b, c and d were performed in cell-attached configuration with Na+ as the only permeating cation in the recording pipette.