Molecular mechanisms underlying the pilsicainide-induced stabilization of hERG proteins in transfected mammalian cells

Abstract

Background: Pilsicainide, classified as a relatively selective Na⁺ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K⁺ current (I_Kr ) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system.

Methods: HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments.

Results: Acute exposure to pilsicainide at 0.03-10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03-10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W.

Conclusions: Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

Keywords: Chemical chaperone; Pilsicainide; hERG.

Fig. 1

Acute effects of pilsicainide on the expression of WT-hERG proteins and channel currents. (A) Effects of 10 min application of pilsicainide at 0.03–10 μM on WT-hERG protein expressions. Representative Western blots show bands of hERG proteins at 135 kDa (immature form) and 155 kDa (mature form) with those of β-actin being used for normalizing the band density of hERG proteins (left). The density of the immature and mature forms of hERG proteins determined at each concentration of the agent was normalized first to that of β-actin and then to that of WT-hERG-FLAG in the absence of pilsicainide (right; n=4 each). (B) Effects of 10 min application of pilsicainide on hERG channel currents. Shown are representative traces of E4031-sensitive hERG channel currents recorded in HEK293 cells expressing WT-hERG-FLAG in the absence (Control) and presence of pilsicainide at 3 μM (left). Current–voltage relationships were determined for WT-hERG-FLAG protein-mediated peak currents during the depolarizing test pulses (right; n=10 each). Error bars smaller than the radius of the circles are not shown.

Fig. 2

Chronic effects of pilsicainide on the expression of WT-hERG proteins and channel currents. (A) Representative Western blots for WT-hERG-FLAG proteins expressed with or without pilsicainide (left). Cells stably expressing WT-hERG were exposed to pilsicainide at 0.03–10 μM for 48 h. Cell lysates were subjected to Western blotting with anti-FLAG and anti-β-actin antibodies. Two bands for the mature form of 155 kDa and immature form of 135 kDa were observed. The band density of WT-hERG-FLAG proteins was quantified as a ratio to that of β-actin, and was then normalized to that of WT-hERG-FLAG in the absence of pilsicainide (right). Differences were tested for statistical significance by two-way ANOVA: *P<0.05 vs. Control (0) (n=10 each). (B) Representative traces of E4031-sensesitive hERG channel currents recorded from HEK293 cells treated with or without pilsicainide at 3 μM for 48 h (left). Current-voltage relationships were determined for WT-hERG-FLAG protein-mediated peak currents during the depolarizing test pulses in the absence and presence of pilsicainide (right; n=10 each). Differences were tested for statistical significance by two-way ANOVA: *P<0.05 vs. Control. Error bars smaller than the radius of the circles are not shown.

Fig. 3

Chronic effects of pilsicainide on the degradation of WT-hERG proteins. (A) Representative Western blots of residual WT-hERG proteins at 0–24 h after treatment with cycloheximide in the absence (Control) and presence of pilsicainide. Cells in the pilsicainide-treated group were exposed to 3 μM pilsicainide for 48 h prior to the treatment with cycloheximide. (B) Decay of WT-hERG proteins in the absence (Control) and presence of 3 μM pilsicainide. The amount of expressed hERG-FLAG was quantified as a ratio to that of β-actin, and was then normalized to that at time=0. The lines are the fits with a single exponential function. *P<0.05, vs. Control (n=3 each).

Fig. 4

Effects of pilsicainide on the intracellular localization of WT-hHERG-FLAG proteins. Representative immunofluorescence images obtained by a confocal microscope are shown for the WT-hERG-FLAG protein and DsRed2-ER (top), DsRed-Monomer-Golgi (middle) or PM-mKeima-Red (bottom). Cells stably expressing WT-hERG-FLAG proteins were transfected with pDsRed2-ER, pDsRed-Monomer-Golgi or pPM-mKeima-Red, and were stained with anti-FLAG and anti-marker antibodies.

Fig. 5

Effect of membrane-impermeant pilsicainide (N-Me-PIL-MeSO₄) on protein expression of hERG. Representative Western blots are shown for WT-hERG-FLAG proteins expressed in the cells with or without the membrane-impermeant pilsicainide at 0.03–10 μM. Cells stably expressing WT-hERG were exposed to pilsicainide at indicated concentrations for 48 h.

Fig. 6

Effects of pilsicainide on phosphorylation of Akt and expressions of hsp family proteins. (A) Representative Western blots for WT-hERG-FLAG, phosphorylated Akt (p-Akt), total Akt and β-actin in cells treated with or without pilsicainide in the absence and presence of the PI3-kinase inhibitor LY294002. Cells stably expressing WT-hERG were exposed to 3 μM pilsicainide for 48 h. (B) Representative Western blots for WT-hERG-FLAG, HSF-1, hsp70, hsp90 and β-actin in the cells treated with or without pilsicainide. Cells stably expressing WT-hERG were exposed to 3 μM pilsicainide for 48 h.

Fig. 7

Treatment with E4031 abolished the pilsicainide-induced increases in protein expression of WT-hERG. (A) Representative Western blots for WT-hERG-FLAG and β-actin in cells treated with or without pilsicainide in the presence and absence of 10 μM E4031. Cells stably expressing WT-hERG were exposed to 3 μM pilsicainide for 48 h. (B) Summary of the effects of pilsicainide on the mature (155 kDa) and immature (135 kDa) forms of WT-hERG-FLAG proteins in the presence of absence of 10 μM E4031. The amount of expressed WT-hERG-FLAG proteins was quantified as a ratio to that of β-actin, and was then normalized to that of WT-hERG-FLAG in the absence of the agents (Control). *P<0.05 vs. Control (n=5 each).

Fig. 8

Concentration-dependent effects of pilsicainide on the protein expression of the hERG mutants G601S and R752W in transfected HEK293 cells. Cells stably expressing G601S or R752W mutants were exposed to pilsicainide at 0.03–10 μM for 48 h. Cell lysates were subjected to Western blotting with the indicated antibodies.