Figure 4.

DAG, but not IP₃, can be synthesized directly from PI(4)P. (A and B) Simultaneous recording of FRETr from the LIBRAvIII IP₃ probe (top) and KCNQ2/3 currents (bottom) in a cell also expressing M₁R and VSP. Unless noted otherwise, KCNQ2/3 currents were activated by depolarization from −60 to +40 mV for 200 ms every 600 ms and quantified by tail currents. Note that the zebrafish VSP is not activated significantly at +40 mV. (A) Decrease in LIBRAvIII FRETr, reflecting IP₃ production, and KCNQ2/3 current, reflecting PIP₂ depletion, upon the application of Oxo-M. (B) KCNQ2/3 currents and VSP were activated by depolarization to +120 mV for 200 ms every 600 ms during the time indicated (bar). Note that KCNQ2/3 activation is close to saturated at +40 mV, and VSP activation leads to a decrease in KCNQ2/3 current. Subsequent Oxo-M application did not elicit a LIBRAvIII FRETr response. (C) Summary of LIBRAvIII responses (amplitude and duration) in experiments as in A and B (n = 6). Cells were grouped by the extent of PIP₂ depletion during VSP as estimated from KCNQ2/3 current inhibition. 0% corresponds to no VSP activation. (D) Summary of experiments as in E and F (n = 10). (E) Simultaneous recording of KCNQ2/3 currents (bottom) and FRETr from C1-YFP/CFP-CAAX (top, reflecting DAG production) in cells also expressing M₁R and VSP. M₁R activation with 10 µM Oxo-M leads to an inhibition of KCNQ2/3 tail currents and an increase in FRETr, indicating PIP₂ depletion and DAG production. (F) Activation of VSP by depolarization to +120 mV for 200 ms every 600 ms (bar) reduces KCNQ2/3 tail currents without increase in FRETr. Subsequent Oxo-M application did elicit a C1-YFP/CFP-CAAX FRETr increase. (G and H) Simulations from the kinetic model reproducing the observed 70% reduction in the LIBRAvIII response (G) by prior VSP activation but no change in C1-domain response (H). VSP_max was 0 for “no VSP,” 0.3 s⁻¹ during “moderate” VSP activation, and 20 s⁻¹ during “strong” VSP activation. Note that moderate VSP reproduces the time course of KCNQ2/3 current inhibition by VSP that was observed experimentally. [LIBRAvIII] = 6 µM; [C1] = 0.5 µM.