The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

Abstract

Aim: Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K(+) channels expressed in HEK293 cells.

Methods: hERG K(+) currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K(+) channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy.

Results: Application of ATV (0.01-30 μmol/L) concentration-dependently decreased hERG K(+) currents with an IC50 of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K(+) currents. Compared with the wild type hERG K(+) channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K(+) currents. Overnight treatment with ATV (0.1-30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane.

Conclusion: ATV directly blocks hERG K(+) channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.

Figure 1

Concentration-dependent inhibition of human ether-a-go-go-related gene (hERG) potassium (K⁺) currents by atazanavir (ATV). (A) Representative currents in the absence, presence (5 min), and following wash-off (15 min) of 10 μmol/L ATV, elicited by the voltage protocol shown in the inset. (B) Representative currents from the same cell in the absence of ATV and during ATV exposure. (C) Concentration-response curve of ATV-induced inhibition of hERG K⁺ currents in HEK293 cells. The concentration-response relationship shows that the IC₅₀ was 5.7±1.8 μmol/L, and the Hill coefficient was 0.3±0.1. Each drug concentration was evaluated on at least five cells.

Figure 2

Effects of atazanavir (ATV) on human ether-a-go-go-related gene (hERG) potassium (K⁺) currents expressed in HEK293 cells. (A) Original traces of hERG K⁺ currents elicited by the pulse protocol shown in the inset under control conditions (left) and 5 min after incubation with 10 μmol/L ATV (right). (B and C) I–V relationships for (B) currents measured at the end of depolarizing steps and (C) tail currents in control cells and in cells exposed to 10 μmol/L ATV (^bP<0.05, ^cP<0.01 vs control, n=6, mean±SD). (D) Peak tail currents were normalized to their respective maximum current amplitude (control and drugs) to illustrate changes in half-maximal activation voltages. (E) Representative current traces showing steady-state inactivation using the pulse protocol (upper panel).

Figure 3

Effects of atazanavir (ATV) on Y652A and F656C human ether-a-go-go-related gene (hERG) potassium (K⁺) channels. (A) Representative WT-hERG and Y652A-hERG currents in the absence and presence of 30 μmol/L ATV. (B) Representative WT-hERG and F656C-hERG K⁺ currents in the absence and presence of 30 μmol/L ATV. (C) Mean fractional block produced by ATV for WT-hERG and Y652A-hERG. (D) Mean fractional block produced by ATV for WT-hERG and Y652A-hERG. ^bP<0.05 vs wild type. n=5 cells per group; WT, wild type.

Figure 4

Atazanavir (ATV) reduced maturation and surface expression of human ether-a-go-go-related gene (hERG) channels. (A) Western blot showing effects of overnight treatment with increasing concentrations of ATV (0.1, 1.0, 10, and 30 μmol/L) on hERG protein transiently expressed in HEK293 cells. (B) Image densities of fully glycosylated 155 kDa and core-glycosylated 135 kDa hERG proteins were quantified as a function of ATV concentrations using a PhosphorImager. All image densities were normalized to the β-actin protein form measured under control conditions. ^bP<0.05, ^cP<0.01 vs control. n=5. (C) Representative current traces under control conditions and after long-term application of ATV at 1 and 30 μmol/L (see Figure 2A for pulse protocol). (D) Peak tail current densities at each concentration normalized to the value under control conditions. ^bP<0.05, ^cP<0.01 compared with control. n=8.

Figure 5

Double immunofluorescence staining of human ether-a-go-go-related gene (hERG) protein and the endoplasmic reticulum (ER) marker protein calreticulin in HEK293 cells incubated in normal (A) or 30 μmol/L ATV-containing medium (B) for 36–48 h.

Figure 6

Western blot analysis of cells expressing human ether-a-go-go-related gene (hERG) Y652A and F656C mutant channels. Cells expressing hERG Y652A and F656C mutant channels were analyzed by Western blot under control conditions and after 36–48 h incubation with ATV (1.0, 10, and 30 μmol/L). ^bP<0.05 compared with control. n=4 per group.