PGE2 inhibits Kv2.2 channels in HEK293T cells.

(A) Representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV under the control condition (black) and subsequently in the presence of 10 μM PGE2 (red) in the same HEK293T cell. PGE2 was applied to the extracellular solution and recordings taken at an interval of 10 s. (B) The time course of the Kv2.2 current inhibition by 10 μM PGE2. (C) PGE2 inhibited Kv2.2 currents in a concentration-dependent manner. n.s., not significant. ***p < 0.001. One-way ANOVA with Bonferroni post hoc test (0.01 μM: n = 7, p = 0.4002; 0.1 μM: n = 8, p = 0.0672; 1 μM: n = 9, p = 0.0002; 10 μM: n = 24, p < 0.0001; 100 μM: n = 17, p < 0.0001). (D) Left, representative Kv2.2 current recordings in response to 200-ms 10-mV depolarizing steps from -80 to +50 mV in the control (top, black) and PGE2-treated (bottom, red) groups. Right, plot of the current-voltage relationship from Left (n = 7 for each data point). *p < 0.05. Two-tailed paired t-test. (E) Plot of Kv2.2 current activation curves in the control (black) and PGE2-treated (red, n = 7 for each data point) groups. (F) Plot of Kv2.2 current inactivation curves in the control (black) and PGE2-treated (red, n = 7 for each data point) groups.

PGE2 inhibits Kv2.2 currents via the EP2/EP4 signaling pathway in HEK293T cells.

(A) Top, RT-PCR showing the mRNA expression of EP1-4 receptors in HEK293T cells. Bottom, statistics of the mRNA expression of EP1-4 receptors in HEK293T cells (n = 4). (B) Representative examples of immunofluorescence images showing expression of EP1-4 receptors in HEK293T cells. Scale bar, 20 μm. (C) SC51089 (the EP1 receptor antagonist), AH6809 (the EP2 receptor antagonist), L798106 (the EP3 receptor antagonist), and AH23848 (the EP4 receptor antagonist) per se did not alter Kv2.2 currents. n.s., not significant (SC51089: n = 8, p = 0.9154; AH6809: n = 10, p = 0.0661; L798106: n = 9, p = 0.5581; AH23848: n = 6, p = 0.8827). (D) Representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV in the presence of SC51089, AH6809, L798106 and AH23848 respectively, and subsequently in the presence of an additional 10 μM PGE2 in the same HEK293T cell. (E) Statistical analysis showing the effect of EP1-4 antagonists on PGE2-induced inhibition of Kv2.2 channels. ****P < 0.001 versus PGE2 alone by a two-tailed unpaired t-test. n.s., not significant. (+SC51089: n = 5, p = 0.3997; +AH6809: n = 10, p < 0.0001; +L798106: n = 6, p = 0.1785; +AH23848: n = 8, p < 0.0001). (F) Left, representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV under the control condition and subsequently in the presence of the EP2 receptor agonist Butaprost in the same HEK293T cell. Right, statistics for the amplitude of Kv2.2 currents from Left using a two-tailed paired t-test (n = 5). *p = 0.0306. (G) Similar to F, but with the EP4 receptor agonist CAY10598 in the extracellular solution (n = 8). ***p = 0.0003.

PGE2 inhibits Kv2.2 currents via the PKA signaling pathway.

(A) Top, representative western blot showing the PKA phosphorylation level in HEK293T cells following treatment with 10 μM PGE2 for time intervals of 2, 5, and 10 minutes. Bottom, statistics from three independent experiments using a one-way ANOVA with Bonferroni post hoc test (2 min: **p = 0.0078; 5 min: **p = 0.002; 10 min: ***p = 0.0002). (B) Top, representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV under the control condition (black) and subsequently in the presence of 10 μM Db-cAMP (red) in the same HEK293T cell. Bottom, statistics for the amplitude of Kv2.2 currents from Top using a two-tailed paired t-test (n = 6, **p = 0.0035). (C) Similar to B, but with Rp-cAMP in the extracellular solution (n = 8, p = 0.3851). (D) Top, representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV in the presence of 10 μM Rp-cAMP (black) and subsequently in the presence of an additional 10 μM PGE2 (red) in the same HEK293T cell. Bottom, statistics for the amplitude of Kv2.2 current from Top using a two-tailed paired t-test (n = 5, p = 0.6425). n.s., not significant. (E) Schematic structural models of Kv2.2 channels indicate the positions of nine potential PKA phosphorylation sites. (F) Statistics for the amplitude of wild-type and various mutant Kv2.2 channel currents, induced by a depolarization pulse from -80 to +40 mV (n = 6-24). ****P < 0.0001 compared to wild-type Kv2.2 by a one-way ANOVA with Bonferroni post hoc test. (G) Left, representative Kv2.2-S448D mutant channel current traces induced by a depolarization pulse from -80 to +40 mV under the control condition (black) and subsequently in the presence of 10 μM PGE2 (red) in the same HEK293T cell. Right, statistics for the amplitude of Kv2.2 current from Left using a two-tailed paired t-test (n = 6, p > 0.9999). n.s., not significant. (H) Similar to G, this section presents data for the Kv2.2S448A mutant channel in HEK293T cells (n = 6, p > 0.9999).

PGE2 inhibits native Kv2.2 channels in INS-1(832/13) cells.

(A) Representative examples of immunofluorescence images showing high expression of Kv2.2 channels in INS-1(832/13) cells. Scale bar, 20 μm. (B) Left, representative IK traces induced by a depolarization pulse from -80 to +40 mV under the control condition (black) and subsequently in the presence of 10 μM PGE2 (red) in the same INS-1(832/13) cell. Right, statistics for the amplitude of IK from Left using a two-tailed paired t-test (n =10, ****P < 0.0001). (C) Left, representative examples of western blot images showing the effects of the two shRNA oligos (KD1-Kv2.2 and KD2-Kv2.2) on Kv2.2 channel surface expression in HEK293T cells transfected with Kv2.2. Right, statistics for cell surface expression of Kv2.2 channels from Left using a two-tailed unpaired t-test. KD1-Kv2.2: n = 7, **p = 0.0066; KD2-Kv2.2: n = 7, ****p < 0.0001; KD1/KD2-Kv2.2: n=3, ***p = 0.0005, compared with scramble. (D) Knockdown of Kv2.2 channels significantly reduced IK amplitude in INS-1(832/13) cells (n = 6, *p < 0.05). (E) Left, representative IK traces induced by a depolarization pulse from -80 to +40 mV under the control condition (black) and subsequently in the presence of 10 μM PGE2 (red) in the same INS-1(832/13) cell transfected with scramble control or KD2-Kv2.2. (F) Knockdown of Kv2.2 channels abrogated the inhibitory effect of PGE2 on IK in INS-1(832/13) cells. Scramble-PGE2: n =11, ****p < 0.0001; KD2-PGE2: n = 6, p = 0.1227; two-tailed paired t-test. (G) Left, representative Kv2.2 current traces induced by a depolarization pulse from -80 to +40 mV under the control condition and subsequently in the presence of the EP2 receptor agonist Butaprost in the same INS-1(832/13) cell. Right, statistics for the amplitude of Kv2.2 currents from Left using a two-tailed paired t-test (n = 10, ****P < 0.0001). (H) Similar to G, but with the EP4 receptor agonist CAY10598 in the extracellular solution (n = 9, ****P < 0.0001).

PGE2 reduces β-cell electrical excitability through Kv2.2 channels.

(A) Left, representative action potential (AP) firings induced by 20 mM glucose under the control condition and subsequently in the presence of 10 μM PGE2 in the same INS-1(832/13) cell. Right, statistics for the AP firing frequency (*p = 0.0385), amplitude (p = 0.9478), and half-width (* p = 0.0101) from A (n = 9). Two-tailed paired t-test. (B) Similar to A, but the INS-1(832/13) cells were transfected with scramble-Kv2.2 (n = 8) (frequency: *p = 0.0285; amplitude: p = 0.6603; half-width: *p = 0.0281. (C) Similar to B, but the INS-1(832/13) cells were transfected with KD2-Kv2.2 (n = 6). n.s., not significant (frequency: p = 0.4564; amplitude: p = 0.1601; half-width: p = 0.3034). (D) Representative immunofluorescence images showing expression of EP1-4 receptors in INS-1(832/13) cells. Scale bar, 20 μm. (E) Representative immunofluorescence images showing expression of EP1-4 receptors in mouse and human islets. Scale bar, 20 μm.

PGE2 reduces β-cell electrical excitability through EP2/4.

(A) Top, representative AP firings induced by 20 mM glucose under the control condition and subsequently in the presence of 20 μM Butaprost in the same INS-1(832/13) cell. Bottom, statistics for the AP firing frequency (*p = 0.0312), amplitude (p = 0.7354), and half-width (p = 0.2067) from Top (n = 6). Two-tailed paired t-test. (B) Similar to A, but in the presence of 20 μM CAY10598(n = 6), AP firing frequency (*p = 0.0291), amplitude (p= 0.2211), half-width (p = 0.0753).

PGE2 inhibits glucose-stimulated insulin secretion (GSIS) through Kv2.2 channels.

(A) Effects of PGE2 on insulin secretion in INS-1(832/13) cells under basal (2.8 mM, 2.8 G) or stimulatory (16.7 mM, 16.7 G) glucose concentrations (n = 3). 2.8G, PGE2: n.s., not significant (p = 0.9497); 16.7G, PGE2: **p = 0.0082. Two-tailed unpaired t-test. (B) Knockdown of Kv2.2 channels reduced GIGS and greatly alleviated the PGE2-induced inhibition of GSIS in INS-1(832/13) cells (n = 3). ****P < 0.0001; n.s., not significant. Two-tailed unpaired t-tests. (C) Top, cartoon of the Kv2.2 targeted gene disruption. Bottom, PCR products generated from wild-type (+/+, 521 bp), heterozygous (+/−, 521 bp and 415 bp), and homozygous knockout (−/−, 415 bp) mice with primers (F1 and R1) specific to the surrounding Kv2.2 gene disruption sequence and one primer (R2) specific to the targeting sequence. NTC, no template control. (D) Representative immunofluorescence images showing expression of Kv2.2 channels in wild-type and Kv2.2-/- knockout mouse pancreatic islet β-cells. Scale bar, 20 μm. (E) Kv2.2 knockout did not alter the body weight of animals. (F) Left, the effect of PGE2 on the glucose tolerance test in Kv2.2−/−, and control animals. N = 4 animals per group. Right, statistics for AUC from Left. ** P = 0.0064. n.s., not significant (p = 0.9684). Two-tailed unpaired t-tests. (G) Statistics for the effect of PGE2 on GSIS (16.7 mM glucose) in isolated islets from Kv2.2−/−, and control animals. N = 5 animals per group. * P = 0.0483, ** P = 0.001. n.s., not significant (p = 0.4766). Two-tailed unpaired t-tests.

PGE2 inhibits mouse pancreatic β-cell electrical excitability through Kv2.2 channels.

(A) Left, representative IK recordings in response to 200-ms depolarization pulses from - 80 to +50 mV in wild-type and Kv2.2-/- knockout mouse pancreatic β-cells. Right, plot of the current-voltage relationship from Left (n = 7 for each data point). (B) Left, representative IK traces induced by a depolarization pulse from -80 to +40 mV under the control condition and subsequently in the presence of 10 μM PGE2 in the same β-cell from wild-type or Kv2.2-/- mice. Right, statistics for the IK amplitude using a two-tailed paired t-test. ****P < 0.0001, n = 17; n.s., not significant (p = 0.6317, n= 12). (C) Left, representative AP firings induced by 20 mM glucose under the control condition and subsequently in the presence of 10 μM PGE2 in the same β-cell within wild-type mouse pancreatic islets. Right, statistics for the AP firing frequency (*p = 0.0288), amplitude (p =0.8589), and half-width (p = 0.0563) from Left (n = 4). Two-tailed paired t-tests. (D) Left, representative AP firings induced by 20 mM glucose under the control condition and subsequently in the presence of 10 μM PGE2 in the same β-cell within Kv2.2-/- mouse pancreatic islets. Right, statistics for the AP firing frequency (p = 0.1536), amplitude (p = 0.1981), and half-width (p = 0.2385) from Left (n =3). Two-tailed paired t-tests.