Regulation of Kv2.1 channel inactivation by phosphatidylinositol 4,5-bisphosphate

Figures

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  Figure 1. Effect of exogenous PIP2 on the activity of Kv2.1 channels. (A) Representative temporal course of the current amplitude at +60 mV before (black) and during (red) current rundown and during 10 μM PIP2 (blue) application. (B) Representative traces recorded with the protocol shown inset at the time points indicated by the numbers in (A). (C) Average normalized current amplitudes after rundown and exposure to 10 μM PIP2 (n = 11, ***p < 0.001). Error bars represent ±SEM.

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  Figure 2. Effect of holding potential (hp) on Kv2.1 current rundown in excised inside-out patches. (A) Kv2.1 current traces recorded with protocols shown on left. (B) Temporal course of Kv2.1 currents after the change of hp from −120 to −80 mV and then back to −120 mV. (C) Voltage-dependence of inactivation of Kv2.1 channels determined under whole-cell (black), inside-out (red), and inside-out in presence of 10 μM PIP2 (blue). Data points are mean ± SEM (n = 7–10).

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  Figure 3. Impact of PIP2 depletion on Kv2.1 currents. (A) Representative current traces recorded at + 60 mV from a holding potential (hp) of −80 mV under whole-cell configuration. Traces are shown before (control) and after application of 1 μM rapamycin. (B) Average peak and end-pulse current inhibition by rapamycin (n = 10). (C) Representative Kv2.1 current traces recorded before (control) and after application of 10 μM acetylcholine. (D) Average peak and end-pulse current inhibition by acetylcholine (n = 8). Error bars represent ± SEM.

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  Figure 4. Effect of PIP2 depletion on the voltage-dependence of activation of Kv2.1 channels. (A,B) Representative Kv2.1 currents traces obtained in response to an activation protocol (inset) before (A) and after application of 1 μM rapamycin (B). (C) Activation curves of Kv2.1 channels determined in control conditions (black) and after cells were perfused with 1 μM rapamycin (red, n = 10). (D) Activation curves of Kv2.1 channels determined in control conditions (black) and after cells were perfused with 10 μM acetylcholine (violet, n = 8). Data points are mean ± SEM.

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  Figure 5. Effect of PIP2 depletion on the voltage-dependence of inactivation of Kv2.1 channels. (A,B) Representative Kv2.1 currents traces obtained with an inactivation protocol (inset) before (A) and after application of 1 μM rapamycin (B). (C) Inactivation curves of Kv2.1 channels determined under control conditions (black) and after cells were perfused with 1 μM rapamycin (red, n = 8). (D) Inactivation curves of Kv2.1 channels determined under control conditions (black) and after cells were perfused with 10 μM acetylcholine (violet, n = 8). Data points are mean ± SEM.

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  Figure 6. Effect of PIP2 depletion on the kinetics of Kv2.1 closed-state inactivation. (A,B) Representative Kv2.1 currents traces obtained with a closed-state inactivation protocol (inset) before (A) and after application of 1 μM rapamycin (B). (C) Temporal course of closed-state inactivation determined in control conditions (black) and after cells were perfused with 1 μM rapamycin (red, n = 9). (D) Temporal course of closed-state inactivation determined in control conditions (black) and after cells were perfused with 10 μM acetylcholine (violet, n = 7). The data points represent the current evoked by the second depolarizing pulse to + 80 mv, relative to the amplitude of the initial depolarizing pulse, plotted against the duration of the conditioning pulse at −40 mV. Data points are mean ± SEM.

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  Figure 7. Effect of PIP2 depletion on the recovery kinetics of Kv2.1 channels from inactivation. (A,B), Representative Kv2.1 current traces recorded with a 3-pulse protocol (inset) before (A) and after application of 1 μM rapamycin (B). (C) Temporal course of recovery from inactivation determined in control conditions (black) and after cells were perfused with 1 μM rapamycin (red, n = 11). (D) Temporal course of recovery from inactivation determined in control conditions (black) and after cells were perfused with 10 μM acetylcholine (violet, n = 8). The data points represent the peak current evoked by the third pulse (P3), normalized to the peak current elicited by the identical first pulse (P1) and plotted as a function of the time interval at the holding potential (−100 mV). Data points are mean ± SEM.

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  Figure 8. Kinetic model and simulated Kv2.1 currents. (A) Kinetic model:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\,{C}_{i}$$\end{document}Ci,\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\,{I}_{i}$$\end{document}Ii, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$O$$\end{document}O represent closed, inactivated and opened states, respectively. Rate constants \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a$$\end{document}a, \documentclass[12pt]{minimal}…