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. 2015 Jun;145(6):529–541. doi: 10.1085/jgp.201411349

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© 2015 Kim et al.

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Figure 6. — Depletion of PI(4)P and PIP₂ accelerates current inactivation of Ca_V2.2 channels. (A) Analysis of plasma membrane fluorescence intensity of PJ and β2e. Left and right images show the plasma membrane distribution of RFP-PJ and β2e-GFP, respectively, before and after rapamycin addition for 2 min. Bottom and right plots indicate the fluorescence intensity of region of interest (ROI) in RFP-PJ –expressing cells and the intensity of ROI in β2-GFP–expressing cells, respectively, before and after rapamycin addition. (B) Summary of plasma membrane fluorescence intensity of the β2e subunit. Plasma membrane fluorescence intensity of β2e subunit for Dead, INPP5E, Sac, and PJ was normalized against the plasma membrane fluorescence intensity before rapamycin addition. For Dead, n = 7; for INPP5E, n = 7; for Sac, n = 8; for PJ, n = 8. ***, P < 0.001, compared with Dead. (C) Current inactivation of Ca_V2.2 channels with various translocatable systems were measured during a 500-ms test pulse at 10 mV. Current traces before (black trace) and after (red trace) rapamycin application are scaled to the peak current amplitude (I₀) and superimposed. Dashed line indicates zero currents. (D) Summary of inactivation of Ca_V2.2 currents before and after rapamycin addition in cells expressing different translocatable dimerization constructs (n = 4–5). *, P < 0.05, compared with Dead. (E) Voltage dependence of normalized steady-state inactivation for Ca_V2.2 channels with Dead (n = 4), Sac (n = 4), INPP5E (n = 5), or PJ (n = 5) after rapamycin addition. (F) Effect of various translocatable systems on inactivation of Ca_V1.3 channels. Ca_V1.3 currents were measured during a 500-ms test pulse to −10 mV. (G) Summary of inactivation of Ca_V1.3 currents before and after rapamycin addition in cells expressing different translocatable dimerization constructs (n = 4–5). ***, P < 0.001, compared with Dead. (H) Voltage dependence of normalized steady-state inactivation for Ca_V1.3 channels with Dead (n = 4), Sac (n = 5), INPP5E (n = 5), or PJ (n = 5) after rapamycin addition. Data are mean ± SEM.