FIGURE 3.

Depolarization increases KCNQ2/Q3 currents through increasing membrane PIP₂ level. A, co-expressed Ci-VSP was activated at depolarization potentials and induced an inhibition of KCNQ2/Q3 currents. Aa shows the current traces elicited by depolarization from −20 mV to +40 mV. Ab shows the comparison of the currents elicited by +40 mV depolarization with (gray) or without (black) Ci-VSP coexpression. B, activation of Ci-VSP antagonized the depolarization-induced potentiation of KCNQ2/Q3 current. Lesser depolarization to −10 mV was used to activate KCNQ2/Q3 currents only, whereas larger depolarization to +40 mV was used to activate Ci-VSP. C, activation of Ci-VSP interrupted but did not cancel the depolarization-induced potentiation of KCNQ2/Q3 currents. The black dotted line was an average representative current trace seen for KCNQ2/Q3 alone under the depolarization (+40 mV). The solid line was the KCNQ2/Q3 currents from oocytes co-expressing Ci-VSP, recorded using the protocol shown above (solid line). The gray dotted line presented KCNQ2/3 currents computed as if the membrane were depolarized to +40 mV rather than 0 mV where the currents were measured. D, summary data of fold-current increases from KCNQ2/Q3 only, and KCNQ2/Q3+Ci-VSP oocytes. The currents were measured 10 min after holding the membrane at +40 mV. E, high K⁺ incubation and depolarization increased PIP and PIP₂ levels. Cellular PIP and PIP₂ levels were measured using thin layer chromatography (TLC). Oocytes were incubated either in ND96 (control) or in ND96K for 15 min (upper panel), or were held either at −80 mV (control) or 0 mV (depolarization) for 15 min (lower panel). Triplicate (upper panel) or duplicate samples (lower panel) from a single experiment are shown. F, summary data for E. The dots in E were quantified and normalized to the control level. Data are summary of three independent experiments. **, p < 0.01.