Article Figures & Data
Figures
- Fig. 1.
Activation of β-arrestin-biased M3D-arr by CNO increases mature hERG expression levels in a concentration- and time-dependent manner. (A) Concentration-dependent effect of CNO on hERG expression (n = 4). (B) Time-dependent effect of 10 µM CNO on hERG expression (n = 3). In both (A) and (B), hERG-HEK cells were transfected with empty pcDNA3 (Ctrl, ○) or HA-tagged M3D-arr plasmid (•). Twenty-four hours after transfection, cells were treated with CNO under various conditions. Whole-cell lysates were collected for Western blot analysis. In each gel, band intensities of the 155-kDa hERG proteins with CNO treatments were normalized to the control and expressed as relative values. Summarized data are presented in the graphs beneath the Western blots. **P < 0.01 versus control (0 µM and 0 hour, respectively).
- Fig. 2.
CNO treatment increases mature hERG expression and IhERG without affecting hERG mRNA level or the activation-voltage relationship of IhERG in M3D-arr expressing cells. hERG-HEK cells were transfected with pcDNA3 or HA-tagged M3D-arr plasmid. Twenty-four hours after transfection, cells were cultured without [control (Ctrl)] or with 10 µM CNO for 24 hours. (A) Effect of CNO treatment on the expression of hERG channels. Band intensities of the 155- and 135-kDa hERG proteins from CNO-treated cells were normalized to respective controls and expressed as relative values in each gel and are summarized beneath the Western blot images (pcDNA3, n = 4; M3D-arr, n = 7). **P < 0.01 versus Ctrl. (B) Effect of CNO on the relative hERG mRNA expression level. Quantitative reverse transcription PCR was conducted using RNA from cells transfected with pcDNA3 or M3D-arr and cultured without (Ctrl) or with CNO (pcDNA3, n = 3; M3D-arr, n = 3). GAPDH was used as a control housekeeping gene. (C) Families of hERG currents elicited using the protocol shown inset in hERG-HEK cells transfected with pcDNA3 or M3D-arr after a 24-hour CNO (10 µM) treatment. (D) The effects of CNO treatment on the activation-voltage relationships of hERG channels in hERG-HEK cells transfected with pcDNA3 (n = 7–11 cells) or M3D-arr plasmid (n = 10–12 cells from four independent trials). **P < 0.01 versus Ctrl for the maximal tail currents.
- Fig. 3.
CNO treatment affects neither Kv1.5 nor EAG channels in M3D-arr expressing cells. (A) Representative Kv1.5 current (IKv.5) or EAG current (IEAG) in respective stable HEK cells transfected with M3D-arr with or without 24-hour CNO (10 µM) treatment. Summarized current amplitudes are shown below the traces. The numbers above the bar graphs indicate the number of cells examined from four independent experiments. (B). Effects of CNO on the expression of Kv1.5 and EAG channel proteins. The relative band intensities of channel proteins in cells treated with CNO are normalized to control and shown beneath the representative Western blot images (n = 4 for Kv1.5, and n = 4 for EAG).
- Fig. 4.
CNO treatment enhances the interaction between M3D-arr and β-arrestin-1. Coimmunoprecipitation experiments illustrating that CNO treatment intensifies the interaction between M3D-arr and β-arrestin-1 (left) but not between M3D-arr and β-arrestin-2 (right). pcDNA3 or HA-tagged M3D-arr transfected hERG-HEK cells were treated with 10 µM CNO for 24 hours. Whole-cell lysates were immunoprecipitated with a goat anti-β-arrestin-1 or mouse anti-β-arrestin-2 antibody. HA-tagged M3D-arr was detected in the immunoprecipitates using an anti-HA primary and appropriate secondary antibodies. Anti-GAPDH was used as a negative control. The difference in background noise between the two panels is due to the different species of antibodies used for immunoprecipitations. M denotes molecular weight marker. WB denotes a Western blot of M3D-arr expression. Similar data were obtained in five independent experiments.
- Fig. 5.
CNO enhances the β-arrestin-1/M3D-arr interaction. Confocal images showing that CNO treatment intensifies the interaction between β-arrestin-1 and M3D-arr (A), but not the interaction between β-arrestin-2 and M3D-arr (B), at the plasma membrane. HA-tagged M3D-arr-transfected hERG-HEK cells grown on glass coverslips were cultured without [control (Ctrl)] or with 10 µM CNO for 24 hours. Cells were fixed and permeabilized. M3D-arr was labeled with a rabbit anti-HA primary antibody and an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody. β-Arrestin-1 or β-arrestin-2 was labeled with a goat anti-β-arrestin-1 or mouse anti-β-arrestin-2 primary antibody and an Alexa Fluor 488-conjugated donkey anti-goat or goat anti-mouse secondary antibody. Cells are shown in differential interference contrast (DIC) images on the left. Intensities of the fluorescence signals of the lines across the representative cells in each condition are quantified by ImageJ and shown in the right panels. Similar data were obtained in four independent experiments.
- Fig. 6.
CNO treatment increases hERG expression through a PKC-independent but Akt-dependent pathway in M3D-arr-transfected hERG-HEK cells. (A) PKC inhibition does not affect the CNO-induced increase in hERG expression. M3D-arr-transfected hERG-HEK cells were cultured without [control (Ctrl)] or with 10 µM CNO for 24 hours in the absence or presence of 50 µM of H7, a PKC inhibitor (n = 9). (B) Akt inhibition abolishes the CNO-induced increase in hERG expression. M3D-arr-transfected hERG-HEK cells were cultured without (Ctrl) or with 10 µM CNO for 24 hours in the absence or presence of 2.5 µM of Akt-I (n = 6). Whole-cell lysates were extracted and assessed using Western blot analysis. Band intensities of the 155-kDa hERG were normalized to the controls and expressed as relative values in each gel and are summarized beneath the Western blot images. **P < 0.01 versus Ctrl.
- Fig. 7.
CNO-mediated M3D-arr activation increases the expression level of hERG as well as phosphorylated Akt (p-Akt). (A) Effect of CNO on hERG, total Akt, and p-Akt in M3D-arr-transfected hERG-HEK cells. Whole-cell lysates were collected 24 hours after culture without [control (Ctrl)] or with 10 µM CNO (n = 5–10). (B) Effect of Akt activator, SC79, on hERG, total Akt, and p-Akt expression in hERG-HEK cells. Whole-cell lysates were extracted from cells treated with dimethylsulfoxide (DMSO) (Ctrl) or 12 µM SC79 (n = 7). In (A) and (B), band intensities of the 155-kDa hERG, Akt, and p-Akt in CNO- or SC79-treated cells were normalized to their respective controls and expressed as relative values in each gel and are summarized beneath the Western blot images. (C) Effect of SC79 on IhERG. Summarized IhERG amplitudes are shown beneath the representative families of IhERG from cells treated with DMSO (Ctrl) or SC79. The numbers above the bar graphs indicate the number of cells examined from four independent experiments. **P < 0.01 versus Ctrl.
- Fig. 8.
CNO treatment increases mature hERG expression and IhERG through PI3K-mediated activation of Akt in M3D-arr-transfected hERG-HEK cells. M3D-arr-transfected hERG-HEK cells were additionally transfected without or with the PI3K inhibitor, PTEN. Cells were then cultured without [control (Ctrl)] or with 10 µM CNO for 24 hours. PTEN blocks the CNO-induced increase in mature hERG and phosphorylated Akt (p-Akt) expression (A) and IhERG (B). For Western blot analysis, band intensities of the 155-kDa hERG and p-Akt from CNO-treated cells were normalized to their respective controls and expressed as relative values in each gel and are summarized beneath the Western blot images (n = 6). For whole-cell patch clamp, summarized IhERG is shown beneath the representative families of IhERG in each condition. The numbers above the bar graphs indicate the number of cells examined from three independent experiments. **P < 0.01 versus Ctrl.
- Fig. 9.
The CNO-mediated increase in IhERG is abolished by inhibition of PIKfyve or Rab11 in M3D-arr-transfected hERG-HEK cells. hERG-HEK cells were transfected with M3D-arr alone or together with Rab11 DN mutant S25N. Twenty-four hours after transfection, cells were treated with CNO (10 µM, 24 hours) in the absence or presence of PIKfyve inhibitor, YM201636 (0.2 µM, in M3D-arr-transfected cells). Cells were then collected for IhERG recordings. The summarized IhERG amplitudes are shown beneath the representative families of IhERG in each condition. The numbers above the bar graphs indicate the number of cells examined from 3 to 5 independent experiments. **P < 0.01 versus control (Ctrl).
- Fig. 10.
CCh increases hERG and native IKr in the presence of H7, and its effects are blocked by Akt-I. (A) Effects of CCh (50 µM, 24 hours) on hERG expression in H7-treated cells in the absence or presence of Akt-I (n = 4). (B) Effects of CCh (50 µM, 24 hours) on IhERG in 50 µM H7-treated cells in the absence or presence of Akt-I (2.5 µM). The numbers above the bar graphs indicate the number of cells examined from four independent experiments. (C) Effects of CCh (50 µM, 24 hours) on Cs+-mediated IKr (IKr-Cs) in H7-treated neonatal rat ventricular myocytes in the absence or presence of Akt-I. The numbers above the bar graphs indicate the number of cells examined from five independent experiments. **P < 0.01 versus control (Ctrl).
- Fig. 11.
Illustration of M3D-arr-mediated β-arrestin signaling in hERG regulation. Activation of M3D-arr by CNO leads to the recruitment of β-arrestin-1, which promotes PI3K-dependent activation of Akt. Enhancement of Akt activity stimulates PIKfyve which promotes Rab11-mediated hERG recycling, leading to an increased mature hERG expression.
