Osmotic stress and PIEZO1 activity. The sensitivity of PIEZO1 expressed in HEK293 cells to different environmental osmolality was analyzed. (A) Changes in cell diameter were measured after solutions of different tonicity were applied to the cell surface; n = 61/Osm, ***P < .001. (B-C) MA currents applied at 6 µm were recorded in the whole-cell patch-clamp configuration, at holding potential −80 mV in HEK293 cells expressing wild-type PIEZO1 (uninduced and induced). Recordings were obtained from isotonic (Iso; 300 mOsm) physiological solution, after perfusion with hypotonic (Hypo; 200 mOsm) solution, and after washout with isotonic (Iso; 300 mOsm) solution. The same experiments were performed only with isotonic (300 mOsm) physiological solution as a control. There was a dramatic increase in the average peak current of MA currents in hypotonic conditions (P < .005, n = 6). To determine the involvement of PIEZO1 on recorded current responses, 30 µM of the inhibitor gadolinium were added to hypotonic solution. The same experiments were performed only with isosmotic (300 mOsm) physiological solution, as a control. A typical response after the addition of gadolinium is shown. (D) MA currents were similarly measured in HEK293 cells expressing HX-associated mutant PIEZO1 (uninduced and induced), with recordings obtained at isotonic (300 mOsm) and hypotonic (200 mOsm) physiological solutions (Table 3).