Molecular determinants of pentamidine-induced hERG trafficking inhibition

Abstract

Pentamidine is an antiprotozoal compound that clinically causes acquired long QT syndrome (acLQTS), which is associated with prolonged QT intervals, tachycardias, and sudden cardiac arrest. Pentamidine delays terminal repolarization in human heart by acutely blocking cardiac inward rectifier currents. At the same time, pentamidine reduces surface expression of the cardiac potassium channel I(Kr)/human ether à-go-go-related gene (hERG). This is unusual in that acLQTS is caused most often by direct block of the cardiac potassium current I(Kr)/hERG. The present study was designed to provide a more complete picture of how hERG surface expression is disrupted by pentamidine at the cellular and molecular levels. Using biochemical and electrophysiological methods, we found that pentamidine exclusively inhibits hERG export from the endoplasmic reticulum to the cell surface in a heterologous expression system as well as in cardiomyocytes. hERG trafficking inhibition could be rescued in the presence of the pharmacological chaperone astemizole. We used rescue experiments in combination with an extensive mutational analysis to locate an interaction site for pentamidine at phenylalanine 656, a crucial residue in the canonical drug binding site of terminally folded hERG. Our data suggest that pentamidine binding to a folding intermediate of hERG arrests channel maturation in a conformational state that cannot be exported from the endoplasmic reticulum. We propose that pentamidine is the founding member of a novel pharmacological entity whose members act as small molecule antichaperones.

Fig. 1.

Pentamidine does not reduce hERG surface expression on short-term exposure. A, Western blot showing time-dependent effects of incubation with 30 μM pentamidine (pent) on hERG stably expressed in HEK293 cells. B, quantitative analysis of time-dependent changes in fg- and cg-hERG levels after exposure to 30 μM pentamidine. Fast effects of 0 K⁺ on fg-hERG (fg, 0K) are due to increased endocytic uptake. Image densities on Western blots were normalized to fg-hERG levels measured at t = 0. Note that cg-hERG is increased at t = 16 to 24 h (n = 3–4). C, representative hERG current families recorded under control conditions, after a 6- or 24-h (overnight) exposure to 30 μM pentamidine. Currents were elicited using depolarizing voltage steps from −60 to +60 mV. Tail currents were recorded on return to −50 mV. Holding potential was −80 mV. D, time-dependent reduction of hERG tail current densities on exposure to 30 μM pentamidine (n = 6–10). Data are given as mean ± S.E.

Fig. 2.

Pentamidine inhibits hERG forward trafficking. A, pulse-chase analysis of hERG maturation in ³⁵S-labeled HEK/hERG cells under control conditions or after overnight incubation with 30 μM pentamidine (pent). Radiolabeled hERG was isolated by immunoprecipitation after chase periods indicated. Arrows indicate position of fg and cg forms of hERG. B, quantitative analysis of time-dependent changes of fg- and cg-hERG densities measured under control (con) conditions or after overnight incubation with 30 μM pentamidine (n = 3–4). C, subcellular localization of hERG protein under control conditions and after overnight exposure to 30 μM pentamidine. HEK/hERG cells were fixed, permeabilized, and double labeled with anti-hERG and anti-KDEL antibody that was used as ER marker. In untreated controls, hERG was localized to the cell surface. In cells treated with pentamidine, hERG staining was no longer detectable at the cell surface. Instead, hERG staining was restricted to the ER. Scale bar, 20 μm.

Fig. 3.

Rescue of pentamidine-induced trafficking inhibition by incubation with the pharmacological chaperone astemizole. A, Western blot showing effects of overnight treatment with increasing concentrations of astemizole (ast) on HEK/hERG cells coincubated with either 10 or 30 μM pentamidine (pent). B, quantitative analysis of concentration-dependent rescue of fg-hERG by astemizole after coincubation with either 10 or 30 μM pentamidine. Astemizole restored hERG trafficking in the presence of 10 μM pentamidine half-maximally at a concentration of 335 ± 33 nM (n = 3). In the presence of 30 μM pentamidine, astemizole rescue was half-maximal at 962 ± 89 nM (n = 3). C, maximal hERG tail current densities measured on return to −50 mV (holding potential was −80 mV; 2-s depolarizing test pulses) under control conditions, on overnight incubation with 30 μM pentamidine, 30 μM pentamidine + 5 μM astemizole (washout, 1 h) and 5 μM astemizole to determine residual block after 1 h washout (n = 6–7). Note that coincubation with 5 μM astemizole significantly increased hERG tail currents after overnight incubation with 30 μM pentamidine. Current densities measured on rescue were not significantly different from densities measured on incubation with astemizole alone.

Fig. 4.

A mutation in the selectivity filter alters sensitivity of hERG to pentamidine induced trafficking inhibition. A, Western blots showing effects of overnight treatment with increasing concentrations of pentamidine on bEAG, hERG WT, hERG F627Y, and hERG S631A transiently expressed in HEK293 cells. B, quantitative analysis of fully glycosylated form of bEAG (n = 3), hERG WT (n = 5–6), hERG F627Y (n = 6), hERGS641A (n = 4), and hERG S631A (n = 3) after long term-exposure to 10 and 30 μM pentamidine (pent). Image densities of fully glycosylated protein bands on Western blots were captured using a Kodak Imager, quantified, and normalized to untreated controls. Black bars indicate a significant difference between fg-WT and bEAG or fg-hERG F627Y on treatment with either 10 or 30 μM pentamidine (Dunnett's t test; p < 0.05). C, hERG WT (n = 7–8), bEAG (n = 6–7) and hERG S631A (7–9) currents were activated in the presence of 5 mM [K⁺]_ex from a holding potential of −80 mV with depolarizing pulses (2 s). Maximal hERG WT tail current amplitudes were recorded on return to −120 mV. BEAG and hERG S631A current amplitudes were recorded at the end of depolarizing pulses to +20 mV. For hERG F627Y (n = 9–7) maximal tail current amplitudes were recorded on return to −80 mV in the presence of 140 mM [K⁺]_ex. Tail current amplitudes were converted into current densities and normalized to amplitudes measured in untreated controls. Black bars indicate a significant difference between WT and bEAG or hERG F627Y current densities on exposure to 30 μM pentamidine (Dunnett's t test; p < 0.05).

Fig. 5.

Mutation of Phe656 in S6 transmembrane helix alters sensitivity to pentamidine-induced trafficking inhibition. A, Western blot showing effects of overnight treatment with increasing concentrations of pentamidine on hERG F656W and hERG F656V transiently expressed in HEK293 cells. B, quantitative analysis of fg form of hERG Phe656 (WT, n = 5–6), hERG 656Trp (n = 5), 656Tyr (n = 4), 656Met (n = 3), 656Thr (n = 4), 656Cys (n = 3), 656Ala (n = 3), and 656Val (n = 4) after overnight treatment with 10 or 30 μM pentamidine. Image densities of fully glycosylated protein bands on Western blots were captured using a Kodak Imager, then quantified and normalized to untreated controls. Black bars indicate a significant difference between fg-Phe656 (WT) and fg-656Thr and 656Val on treatment with 30 μM pentamidine (Dunnett's t test; p < 0.05). C, WT (n = 7–8) or mutant currents (Trp, n = 6–8; Met, n = 7–8; Thr, n = 7–12; Cys, n = 4–6; Ala, n = 6–8; Val, n = 5–7) were activated in the presence of 5 mM [K⁺]_ex from a holding potential of −80 mV with a depolarizing pulse to + 60 mV (2 s). Tail currents were recorded on return to −120 mV. Tail current amplitudes were converted into current densities and normalized to amplitudes measured under control conditions. Black bars indicate a significant difference between WT and F656T, F656C, and F656V on treatment with 30 μM pentamidine (Dunnett's t test; p < 0.05).

Fig. 6.

Long-term exposure to pentamidine does not alter tetrameric structure of hERG. A, sedimentation analysis of heterologously expressed hERG WT channels incubated overnight either in the presence of 1 μg/ml brefeldin A to inhibit complex glycosylation of the channel protein or in a combination of 1 μg/ml brefeldin A and 30 μM pentamidine. Digitonin lysates were separated on 15 to 45% sucrose gradients. Gradient fractions were analyzed by Western blotting using anti-hERG antibody. Arrow indicates the direction of sedimentation. Fraction numbers are indicated below the Western blot. Image densities of gradient fractions were analyzed using a Kodak Imager. B, quantification of steady-state sedimentation gradients as shown in A. Signals from an entire gradient were summed up and set at 100% (total). Individual signals in each fraction were expressed as a percentage of total. P1 and P2 denote two major hERG peaks corresponding to channel monomers/dimers (P1) and channel tetramers (P2) (n = 3).

Fig. 7.

Pentamidine does not interfere with Hsp/c70 and Hsp90 function. A, shown is a pulse-chase experiment performed in the absence and presence of 30 μM pentamidine (pent). Radiolabeled hERG protein was isolated using immunoprecipitation with anti-hERG, anti-Hsp/c70, or anti-Hsp90 antibody after chase periods indicated. Left, positions of fg/cg hERG, Hsp/c70, and Hsp90 on the autoradiogram are indicated. Rightmost lane represents a negative control with no primary antibody added. B, quantitative analysis of time-dependent formation of hERG-Hsp/c70 and hERG-Hsp90 complexes in the absence and presence of pentamidine (n = 3–4). Note that chaperone association is not different between control and pentamidine treated cells.

Fig. 8.

Pentamidine does not increase HERG ubiquitination. Western blot analysis of HEK/hERG cells transiently transfected with HA-tagged ubiquitin (HA-Ub) and treated overnight with either 30 μM pentamidine (Pent) or 10 μM geldanamycin (GA) used as positive control. Transfection with His₆-ubiquitin was used as negative control. Whole-cell lysates were immunoprecipitated with anti-hERG antibody, resolved by SDS-PAGE, and immunoblotted (IB) using either anti-hERG antibody (hERG-IP) or HA-Ub. Immunoblotting with anti-HA antibody identifies high-molecular-weight forms of ubiquitinated hERG that are increased on treatment with geldanamycin but not pentamidine.

Fig. 9.

Pentamidine suppresses endogenous ERG/I_Kr currents in HL-1 cardiomyocytes and in NRVMs. A, Western blot showing mERG in HL-1 cardiomyocytes under control conditions and after overnight exposure to either 1 or 10 μM pentamidine (pent). Note that both mERG1A and mERG1B were reduced by pentamidine, whereas mKv1.5 was not affected. Actin was used as loading control. B, mERG currents recorded in HL-1 cardiomyocytes under control conditions or after overnight exposure to 10 μM pentamidine. Currents were elicited using 2-s depolarizing test pulses from −60 to +60 mV. Holding potential was −80 mV. Tail currents were recorded on return to −50 mV. C, quantitative analysis of maximal mERG tail current densities under control conditions or after overnight exposure to 10 μM pentamidine (n = 6–7). Data are represented in statistical box blots. Note that current densities were significantly different at p < 0.05 level. D, Western blot showing rERG in NRVMs under control conditions and after overnight exposure to 30 μM pentamidine, to 30 μM pentamidine + 3 μM astemizole (Ast), and to 3 μM astemizole alone. Note that coincubation with astemizole rescued fg-rERG in the presence of pentamidine. E, rERG currents in NRVMs were elicited in symmetric Cs⁺ solutions using depolarizing test pulses from −60 to +60 mV. Holding potential was −80 mV. F, quantitative analysis of maximal rERG tail current densities recorded on return to −80mV under control conditions or after overnight exposure to 30 μM pentamidine (n = 5). Data are given as mean ± S.E. Note that current densities were significantly different at p < 0.05 level.