Figure 3.
Figure 3. DiC8-PIP2 slows activation and enhances mode shift in Elk1 in excised patches. (A; left) Currents recorded in response to a 2-s voltage ramp (−120 to 80 mV; −100-mV holding potential) before and after the application of 10 µM PIP2. (Middle and right) Currents recorded in response to a series of 1-s voltage steps (−120 to 60 mV in 20-mV increments; −100-mV holding potential) before and after the application of 10 µM PIP2, respectively. Arrows point to slowed activation to deactivation in the PIP2 traces. (B) The application of 10 µM PIP2 shifts Elk1 G-V in the depolarized direction (n = 37). On-cell and excised data are the same as in Fig. 1 D. Curves are single Boltzmann fits, and V50 and s are reported in Table 1. (C) Normalized current traces from an Elk1 patch comparing the activation rate at −20 mV on cell, excised, and with 10 µM PIP2. Traces were reversed in polarity for display. (D) Change in activation rate upon patch excision and patch application of 10 µM PIP2 at various voltages expressed as τact(on cell)/τact(ctrl) and τact(PIP2)/τact(ctrl) (n = 33 and 18, respectively). (E) Normalized tail currents recorded at −100 mV after 2-s prepulse to 60 mV before and after the application of 10 µM PIP2 (dotted line, zero current). (F) Fractional tail current remaining 60 ms after repolarization to −100 mV in 10 µM PIP2 (n = 14; curve is a single-exponential fit with a time constant of 611 ms) compared with excised and on cell from Fig. 2 E. (G) G-V curves determined from isochronal tail currents at −100 mV after 1-s steps to the indicated voltages for excised controls (red; n = 19) and 10 µM PIP2 (blue; n = 18) from a holding potential of −100 mV (squares) or with a 1-s prepulse to 60 mV (circles). Mode shift values in millivolts are reported in Table 2. (H–J) Dose–response relationships for PIP2 as measured by τact-PIP2/τact-ctrl at −20 mV (H; n = 5–20), V50 shift (I; n = 14–26), and G-V mode shift size (J; ΔV50, −100-mV hold vs. 60-mV prepulse; n = 4–10). Curves show fits with the Hill equation, and half-maximal concentrations (K1/2) are indicated. All data are expressed as mean ± SEM.

DiC8-PIP2 slows activation and enhances mode shift in Elk1 in excised patches. (A; left) Currents recorded in response to a 2-s voltage ramp (−120 to 80 mV; −100-mV holding potential) before and after the application of 10 µM PIP2. (Middle and right) Currents recorded in response to a series of 1-s voltage steps (−120 to 60 mV in 20-mV increments; −100-mV holding potential) before and after the application of 10 µM PIP2, respectively. Arrows point to slowed activation to deactivation in the PIP2 traces. (B) The application of 10 µM PIP2 shifts Elk1 G-V in the depolarized direction (n = 37). On-cell and excised data are the same as in Fig. 1 D. Curves are single Boltzmann fits, and V50 and s are reported in Table 1. (C) Normalized current traces from an Elk1 patch comparing the activation rate at −20 mV on cell, excised, and with 10 µM PIP2. Traces were reversed in polarity for display. (D) Change in activation rate upon patch excision and patch application of 10 µM PIP2 at various voltages expressed as τact(on cell)/τact(ctrl) and τact(PIP2)/τact(ctrl) (n = 33 and 18, respectively). (E) Normalized tail currents recorded at −100 mV after 2-s prepulse to 60 mV before and after the application of 10 µM PIP2 (dotted line, zero current). (F) Fractional tail current remaining 60 ms after repolarization to −100 mV in 10 µM PIP2 (n = 14; curve is a single-exponential fit with a time constant of 611 ms) compared with excised and on cell from Fig. 2 E. (G) G-V curves determined from isochronal tail currents at −100 mV after 1-s steps to the indicated voltages for excised controls (red; n = 19) and 10 µM PIP2 (blue; n = 18) from a holding potential of −100 mV (squares) or with a 1-s prepulse to 60 mV (circles). Mode shift values in millivolts are reported in Table 2. (H–J) Dose–response relationships for PIP2 as measured by τact-PIP2/τact-ctrl at −20 mV (H; n = 5–20), V50 shift (I; n = 14–26), and G-V mode shift size (J; ΔV50, −100-mV hold vs. 60-mV prepulse; n = 4–10). Curves show fits with the Hill equation, and half-maximal concentrations (K1/2) are indicated. All data are expressed as mean ± SEM.

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