Volume 22, Issue 2, Pages 471-481 (January 2018) Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells  Young-Eun Han, Jung Nyeo Chun, Min Jeong Kwon, Young-Sun Ji, Myong-Ho Jeong, Hye-Hyun Kim, Sun-Hyun Park, Jong Cheol Rah, Jong-Sun Kang, Suk-Ho Lee, Won-Kyung Ho  Cell Reports  Volume 22, Issue 2, Pages 471-481 (January 2018) DOI: 10.1016/j.celrep.2017.12.049 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Effects of High-Glucose Stimulation on the Subcellular Distribution of KATP Channels Are Dynamin Dependent (A) Confocal images of INS-I cells stained with Alexa-488-conjugated donkey anti-rabbit IgG antibody (1:100; Invitrogen) after overnight incubation with anti-Kir6.2 antibodies (H-55; sc-20809; 1:50; Santa Cruz Biotechnology) at 4°C after glucose deprivation (GD) under conditions of 0 mM glucose for 2 hr or varying time of incubation in 17 mM glucose solutions (HG) with or without dynasore (Dyn) (25 μM) for 5 min or 30 min. The scale bar represents 5 μm. (B) Western blot analysis using anti-SUR1 and anti-Kir6.2 antibodies to quantify changes in surface KATP channel levels after HG stimulation with or without dynasore. Antibodies to Na+/K+ pump (1:1,000; Abcam) were used as loading control for surface proteins. (C and D) The relative ratios of surface to total SUR1 (C) and that of Kir6.2 (D) were obtained based on the quantification of band intensities and normalized to the value obtained in GD. The data are expressed as the mean ± SEM (n = 8 for SUR1-S in control; n = 4 for other conditions); ∗∗∗p < 0.001 compared with GD using ANOVA with a Bonferroni’s post hoc test. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Effects of Inhibiting Endocytosis on Glucose-Induced Changes in Membrane Potentials and Whole-Cell Conductance in INS-1 Cells (A–D) Representative traces of RMP changes induced by HG stimulation in the control conditions (A) or in the presence of dynasore (B) and in cells transfected with DNM1-WT (C) or DNM1-K44A (D). Dotted lines (gray) indicate the level of −70 mV. (E and F) Representative traces for changes in whole-cell currents induced by HG stimulation in the control (E) and in the presence of dynasore (F). Membrane potential was held at −70 mV, and step pulses (depolarization followed by hyperpolarization with 500-ms duration and 20-mV amplitude) were applied every 10 s. (G) Bar graphs summarize pooled data of RMP obtained under the conditions as indicated (mean ± SEM; n = 13, 13, 5, and 4 for control, dynasore, DNM1-WT, and DNM1-K44A, respectively). ∗∗∗p < 0.001, ∗p < 0.05, and ∗∗∗p < 0.001 compared with GD, GD+Dyn, and GD+DNM1-K44A using paired t test, respectively. (H) Bar graphs summarize mean values for normalized conductance in the absence and presence of Dyn as indicated (mean ± SEM; n = 7 and 13 for control and dynasore, respectively). ∗∗∗p < 0.001 compared with GD and GD+Dyn using paired t test, respectively. (I) Relative concentration of intracellular ATP of INS-1 cells incubated for 2 hr in 0 mM glucose (GD) and that for 30 min in HG in the absence (mean ± SEM; from 4 independent experiments) and presence of dynasore (mean ± SEM; from 3 independent experiments). GD was arbitrarily set at 100%. ∗∗p < 0.01 and #p < 0.05 compared with GD and GD+Dyn using ANOVA, respectively. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Effects of High-Glucose Stimulation on Membrane Potentials and GKATP in INS-1 Cells (A) Representative traces for voltage responses induced by a constant current injection applied every 10 s to measure changes in membrane conductance (Gin) during membrane depolarization by HG (left) or by inhibiting KATP channels (middle) and hyperpolarization by activating KATP channels by DNP (right). Dotted line (gray) indicates the level of −70 mV. (B) Membrane potentials obtained during the application of HG (black), tolbutamide (Tol) (red), or DNP (blue) were plotted against corresponding Gin values (each symbol represents data from one cell). Black line is the theoretical relationship between membrane potentials (RMP) and Gin obtained from the equation RMP = −75 × (Gin − 0.02)/Gin. (C) Bar graphs summarize mean values for GKATP (nS/pF) under the conditions indicated (mean ± SEM; n = 13, 17, 9, and 6 for DNP, GD, HG, and Tolb, respectively). ∗∗∗p < 0.001 compared with GD using ANOVA. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Relative Contribution of Changing KATP Channel Density and Gating to the Changes in GKATP and Vm by HG (A) Representative traces for voltage responses induced by a constant current injection (10 pA) applied every 10 s to measure changes in membrane conductance (Gin) during membrane depolarization by HG. The voltage responses marked in black (a) and red (b) boxes are averaged and displayed in an expanded timescale. (B) Representative traces for voltage responses induced by a 10-pA current injection applied every 10 s to measure changes in membrane conductance during diazoxide application in HG. The voltage responses marked in black (a) and red (b) boxes are averaged and displayed in an expanded timescale. (C) Averaged values of RMP and GKATP obtained under different experimental conditions were plotted on the theoretical relationship between RMP versus GKATP obtained from the equation RMP = −75 × GKATP/(GKATP + 0.02). Triangles represent the contribution of changing KATP channel density (red) and gating (blue) to HG responses. Arrows indicate the effect of maximizing channel opening (blue) and channel number (red). Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Analysis of the Effects of Changing [ATP]i and Surface Density of KATP Channels on GKATP and Vm (A and B) Relationship between [ATP]i and GKATP in different densities of surface KATP channels. Black line is obtained from the equation GKATP = GKmax/(1 + ([ATP]i/156)ˆ1.1), where GKmax = 3.2 nS/pF (A) and 0.45 nS/pF (B). Using RMP = −75 × GKATP/(GKATP + 0.02), Vm values were obtained from GKAT, and plotted against [ATP]i. (C) Effects of changing GKmax on GKATP (left) and Vm (right) curves are illustrated with actual values for GKATP and Vm obtained in GD, HG, and HG + Dyn. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Effects of Inhibiting PKC on Glucose-Induced Decrease in Surface KATP Channels and Membrane Depolarization (A) Representative confocal images of INS-I cells stained with anti-Kir6.2 antibodies after incubation in HG for 20 min in the presence of BIM-I (PKC inhibitor) or BIM-V (inactive analog of BIM-I). The scale bar represents 5 μm. (B) Western blot analysis using anti-SUR1 antibodies to quantify changes in surface KATP channel levels after HG stimulation in the control condition and in presence of BIM-I or BIM-V. (C) Representative traces of RMP changes by HG in INS-1 cells in the presence of BIM-I (left) and BIM-V (right). (D–F) Bar graphs summarize pooled data (mean ± SEM) of relative density of SUR1-S (D; 3 independent experiments), RMP (E; n = 5), and [ATP]i (F; 3 independent experiments) under the conditions as indicated. (D) GD was arbitrary set at 1. ∗∗p < 0.01 compared with GD+BIM-V using ANOVA. (E) ∗∗p < 0.01 compared with GD+BIM-V using paired t test. (F) GD was arbitrary set at 100%. ∗∗p < 0.01 compared with GD+BIM-I using ANOVA. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions

Figure 7 A Model for Illustrating the Difference between ATP Sensitivity of GKATP and that of Vm Changes ATP-dependent changes in Vm (red lines) obtained under a given ATP-inhibition curve for GKATP (black line) are determined by GKmax/GL ratio. Cell Reports 2018 22, 471-481DOI: (10.1016/j.celrep.2017.12.049) Copyright © 2017 The Authors Terms and Conditions