Figure 3: Attenuated maximal current responses in heterologous cells expressing PIEZO1-G2029R.

Wild-type hPIEZO1 and hPIEZO1-G2029R were expressed in HEK-P1KO cells. (a) Fluorescent calcium imaging of cells loaded with Fura-2 expressing PIEZO1 or PIEZO1-G2029R constructs, both containing EGFP driven by an Internal Ribosomal Entry Site for transfection control. Average fluorescence ratios (340 nm/380 nm) of GFP-positive cells are plotted, normalized to baseline ratio values. (b) Representative whole-cell patch-clamp recordings performed using a blunt-ended mechanical probe, which was moved at 1-μm increments to elicit mechanically induced PIEZO1 and PIEZO1-G2029R current responses. A schematic of the measurement configuration is depicted in the inset. Dashed lines indicate zero current levels in the individual traces. A recording of the displacement steps of the mechanical probe is shown above each trace, with the first step that induced visible contact between the probe and the cell depicted in blue colour. Horizontal scale bars, 25 ms, vertical scale bars, 1 nA. (c) Current–voltage relationships recorded at the 3rd mechano-elicited response show no differences between wild-type and G2029R channels (n=5). Insets show representative recordings for wild-type (black) and G2029R (red) channels. Reversal potential was 3.4±1.08 mV and 3.83±0.45 mV for wild-type and G2029R channels, respectively. Horizontal scale bars, 10 ms, vertical scale bars, 0.1 nA (d) Analysis of mechanically induced current traces shows strongly decreased maximal current densities in the PIEZO1-G2029R channel. Statistics are shown for the mechano-stimulation step 8 μm past where the probe made visible contact (left). Analysis of inactivation and activation kinetics (centre and right, respectively) of the 3rd mechanically induced response (that is, 2 μm past the mechanical threshold) revealed no differences. n=9 and 12 for wild-type and G2029R channels, respectively. Whiskers in the boxplot depict 1.5 interquartile range, star marks indicate range. Statistical analysis was performed using the Mann–Whitney U-test. At the 0.05 level, only current densities were significantly different (P=0.0023).