Fig. 1.

PIP₂ depletion reduces Kv7.4 currents. A Examples of whole cell K⁺ currents from HEK293 Kv7.4 cells evoked by step depolarisation from −60 mV to +20 mV in the absence (a) and presence of 20 μM wortmannin (b). Currents were recorded every 15 s and wortmannin applied after 60 s. Initial current trace is shown in black in both subpanels. Subsequent traces after 5-, 10- and 20-min intervals are shown in blue, green and red, correspondingly. Subpanel c shows the mean amplitude of K⁺ current at +20 mV in the absence (black) and presence of wortmannin (red). Each point is the mean ± s.e.m. of four cells. B Representative traces of Kv7.4 currents evoked by steps from −60 mV to a range of potential (−90 to +40 mV) in control (a) and after depletion of PIP₂ by the cells preincubation with wortmannin + short (≤30 s) application of trypsin (b). The mean data are shown in subpanel c with control (black, n = 22), wortmannin alone (green, n = 34), wortmannin plus trypsin (red, n = 36) and linopirdine (purple, n = 14). C Example of cell-attached patch recording from HEK293 Kv7.4 cell showing the effect of 20 μM wortmannin. Long-term trace is shown in subpanel a. Representative expanded 1.75-s segments of channel openings taken from subpanel a highlighting channel activity in the absence, and the presence of wortmannin are shown in subpanels b and c. Closed state and multiple open states are denoted by C and O1–O6. D In-cell Western analysis showing that wortmannin, and other known PIP₂ inhibitors, decrease global PIP₂ level in HEK293 Kv7.4 cells (n = 12–23)