Drug-induced long QT syndrome: hERG K+ channel block and disruption of protein trafficking by fluoxetine and norfluoxetine

Abstract

Background and purpose: Fluoxetine (Prozac) is a widely prescribed drug in adults and children, and it has an active metabolite, norfluoxetine, with a prolonged elimination time. Although uncommon, Prozac causes QT interval prolongation and arrhythmias; a patient who took an overdose of Prozac exhibited a prolonged QT interval (QTc 625 msec). We looked for possible mechanisms underlying this clinical finding by analysing the effects of fluoxetine and norfluoxetine on ion channels in vitro.

Experimental approach: We studied the effects of fluoxetine and norfluoxetine on the electrophysiology and cellular trafficking of hERG K+ and SCN5A Na+ channels heterologously expressed in HEK293 cells.

Key results: Voltage clamp analyses employing square pulse or ventricular action potential waveform protocols showed that fluoxetine and norfluoxetine caused direct, concentration-dependent, block of hERG current (IhERG). Biochemical studies showed that both compounds also caused concentration-dependent reductions in the trafficking of hERG channel protein into the cell surface membrane. Fluoxetine had no effect on SCN5A channel or HEK293 cell endogenous current. Mutations in the hERG channel drug binding domain reduced fluoxetine block of IhERG but did not alter fluoxetine's effect on hERG channel protein trafficking.

Conclusions and implications: Our findings show that both fluoxetine and norfluoxetine at similar concentrations selectively reduce IhERG by two mechanisms, (1) direct channel block, and (2) indirectly by disrupting channel protein trafficking. These two effects are not mediated by a single drug binding site. Our findings add complexity to understanding the mechanisms that cause drug-induced long QT syndrome.

Figure 1

Serial dilution densitometry. (a) Typical Western blot of serial dilutions of hERG protein lysates. (b) Analysis of the normalized 155 kDa band densities of Western blots (n=10). Individual normalized densitometry values (mean±s.e.m.) are given in the table (inset).

Figure 2

ECG tracings (normal standard calibration) of leads III and V6 of the patient with Prozac overdose.

Figure 3

Effect of fluoxetine and norfluoxetine on I_hERG. (a and b) Square pulse protocol along with families of I_hERG traces for control conditions and after 1 μM fluoxetine or 3 μM norfluoxetine. (c and d) Action potential waveform protocol along with I_hERG traces for control conditions, after 0.3 and 1 μM fluoxetine or 1 and 3 μM norfluoxetine.

Figure 4

(a) Western blot analysis of hERG WT channel protein with increasing concentrations of fluoxetine, norfluoxetine, E4031 and cisapride. (b) I_hERG traces (square pulse protocol, see Figure 3a) for control conditions (left panel), following 24 h incubation without drug washout or after 1 h drug-free conditions for 10 or 30 μM fluoxetine (middle panels), and I_hERG 1 h following washout of 5 μM E4031 (right panel).

Figure 5

(a) Western blot analysis of the time-dependence of the development and recovery of fluoxetine-induced disruption of hERG protein trafficking. (b) Families of I_hERG traces for control conditions, and at different times of incubation in 30 μM fluoxetine, and after removal of fluoxetine from the culture medium.

Figure 6

(a) Families of I_Na recorded for control conditions and following 24 h incubation in 30 μM fluoxetine. (b) I_Na recordings for control conditions and after 10 min superfusion of 30 μM fluoxetine. (c) I–V plots of I_Endogenous for control conditions and after 24 h incubation in 30 μM fluoxetine. Insets show representative families of current traces.

Figure 7

(a and b) Concentration–response relations for fluoxetine and norfluoxetine, obtained using square pulse protocol, action potential waveform protocol and from normalized Western blot image density measurements fitted to Hill equation (see text for detail).

Figure 8

(a) Western blot analysis of F656A and F656C mutations for control conditions and following 24 h incubation with 1 and 30 μM fluoxetine. (b) Plots of normalized peak tail I_hERG (square pulse protocol) for WT, and the F656A and F656C mutations, for control conditions and following 1 or 30 μM fluoxetine exposure.