Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome

Abstract

Background and purpose: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein.

Experimental approach: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry.

Key results: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking.

Conclusions and implications: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.

Figure 1

Current–voltage relationship for hERG channels and ketoconazole block. (a) hERG currents under control conditions and in the presence of 4 μM ketoconazole were recorded using the pulse protocol shown. (b, c) Normalized (to control values) I–V relationships for current measured at the end of depolarizing steps (b) and tail currents (c) in control and 4 μM ketoconazole-treated cells (n=6). (d) Peak tail currents were normalized to their respective maximum current amplitude (control and drugs) to illustrate changes in half-maximal activation voltages. Solid lines represent fits to Boltzmann function. Ketoconazole neither changed the half-activation voltage nor the slope factor (V_1/2: –11.6±2.2 and –14.7±2.5 mV; κ. 8.2±0.4 and 8.4±0.5; n=6, in controls and after ketoconazole, respectively; P>0.05). Data are mean±s.e.m.

Figure 2

Ketoconazole inhibits hERG current by accessing the Y652 and F656 amino-acid residues. (a) Representative current traces under control conditions and after ketoconazole treatment (40 μM; 1st, 2nd, 3rd, 4th and 20th current traces are shown). (b) Time course of ketoconazole-induced hERG peak tail current inhibition from the same cell as in panel a. (c) Concentration–response relationships for peak tail current in hERG wild-type channels, as well as in Y652A and F656C mutants. Currents in the presence of ketoconazole were normalized to the control amplitudes and plotted as a function of drug concentration. Lines represent fits with the Hill equation. Data are mean±s.e.m. The numbers by each symbol represent the number of cells tested. (d) Comparison of hERG current inhibition by 40 μM ketoconazole with wild-type (n=5), Y652A (n=6) and F656C (n=5) mutant channels. The hERG currents evaluated at peak tail current amplitude in the presence of ketoconazole were normalized to respective control values. Data are mean±s.e.m. ^*P<0.05, compared with the wild-type. #P<0.05, Y652A vs F656C.

Figure 3

Ketoconazole reduced maturation and surface expression of hERG channels. Concentration-dependent decrease of 155-kDa hERG protein (a), fluorescence intensity of antibody-labelled hERG channels detected by flow cytometry (b) and current densities (c). The cells were incubated for 48 h in control or ketoconazole-containing medium. Panel a, upper panel: representative western blot analysis. Data are mean±s.e.m. ^*P<0.05, compared with the control (n=6). Panel b, data are mean±s.e.m. ^*P<0.05, compared with the control (n=9). Panel c, upper lane: representative current traces under control conditions and after long-term application of ketoconazole at 1 and 10 μM (see Figure 2a for pulse protocol). The hERG current density was measured after 1 h following the washing of the drug. Peak tail current densities at each concentration were normalized to the value in control conditions. Data are mean±s.e.m. ^*P<0.05, compared with the control (n=16–21). (d) Time course of direct hERG current block by 1 μM ketoconazole after 48-h incubation with 1 μM ketoconazole. Amplitude of the peak tail current was plotted against time (see Figure 2a for pulse protocol). An acute hERG current block occurred after disruption of hERG protein trafficking.

Figure 4

Acute and chronic effects of ketoconazole on the hNa_v 1.5 Na⁺ channel. (a) Time course of peak inward Na⁺ current amplitude during ketoconazole (10 μM) wash-in protocol. Representative current recordings indicated by asterisks are shown in the box. (b) Upper lane: representative Na⁺ current traces under control conditions and after cell incubation with 10 μM ketoconazole for 48 h. The Na⁺ current density was measured after 1 h following the washing of the drug. The Na⁺ current was elicited by a 24-ms depolarization step to –20 mV from a holding potential of –150 mV. Ketoconazole treatment did not significantly alter the current density of the peak Na⁺ current. Data are mean±s.e.m. (n=13 in controls and n=16 after ketoconazole).

Figure 5

(a) Western blotting analysis of the development (left) and recovery (right) of 30 μM ketoconazole-induced disruption of hERG protein trafficking over time. Data are mean±s.e.m. ^*P<0.05, compared with the control, #P<0.05, compared with just after washing of the drug (recovery 0), 2, 4, 8 and 24 h (n=4–6). (b) Western blotting of cells expressing hERG F656C and Y652A mutant channels under control conditions, and following 48 h incubation with 1–30 μM ketoconazole. Data are mean±s.e.m. ^*P<0.05, compared with the control (n=6).