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Am J Physiol Heart Circ Physiol 287: H652-H658, 2004. First published April 8, 2004; doi:10.1152/ajpheart.00052.2004
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Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits

Qiuming Gong,1 Corey L. Anderson,2 Craig T. January,2 and Zhengfeng Zhou1

1Division of Molecular Medicine, Department of Medicine, Oregon Health and Science University, Portland, Oregon 97239; and 2Departments of Medicine (Cardiology) and Physiology, University of Wisconsin, Madison, Wisconsin 53792

Submitted 21 January 2004 ; accepted in final form 6 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1) to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2) to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies.

ion channels; arrhythmia; protein trafficking; patch clamp

LONG QT SYNDROME (LQTS) is a disease characterized by delayed cardiac repolarization and prolonged QT intervals on the electrocardiogram and can lead to syncope and life-threatening ventricular arrhythmias (12). The principal form of LQTS, Romano-Ward syndrome, is now recognized as genetically heterogeneous involving multiple genes, and it follows an autosomal dominant inheritance pattern with variable penetrance (16, 25). The chromosome 7-linked form, LQT2, is caused by mutations in the human ether-a-go-go-related gene (HERG or KCNH2) (3). HERG encodes the pore of the rapidly activating component of the delayed rectifier K+ channel current (IKr), which is one of the major ion channel currents contributing to repolarization of the cardiac action potential (23, 27). The mechanisms of HERG channel dysfunction in LQT2 have been studied by expressing disease-causing mutations in Xenopus oocytes or in mammalian cell lines, and with these methods multiple mechanisms were identified including defective protein trafficking, abnormal gating or permeation, and dominant negative suppression of wild-type (WT) HERG channel function (9). Furthermore, we previously showed that some LQT2 mutations, including N470D, exhibit temperature-sensitive protein trafficking defects (29). When expressed alone as a homomeric channel, the N470D mutant is trafficking deficient at 37°C with immature protein retained in the endoplasmic reticulum (ER), whereas at 27°C its trafficking to the plasma membrane and current generation are markedly improved. We also showed that HERG channel blocking drugs could correct (pharmacologically rescue) the trafficking defective N470D mutant channel.

As with other voltage-gated K+ channels, the HERG channel is thought to form a tetrameric subunit structure. Because LQT2 follows an autosomal dominant inheritance pattern, normal and mutant alleles are present in affected patients. Thus mutant subunits may interact with WT subunits to form heteromeric channels, and this may lead to a dominant negative effect to cause a marked loss of current phenotype (see Ref. 9 for a discussion). In fact, when coexpressed with WT subunits in Xenopus oocytes, the N470D mutant causes dominant negative suppression of WT HERG current (22). For other LQT2 mutants, several mechanisms responsible for the dominant negative effect have been reported and include 1) mutant subunits coassemble with WT subunits to alter heteromeric channel function in the cell membrane (18, 19, 22), 2) mutant subunits coassemble with WT subunits to disrupt normal protein trafficking and reduce surface membrane expression of the heteromeric channel (4), and 3) mutant subunits reduce WT HERG protein expression (10). In these studies, a marked reduction in HERG current with WT-LQT2 mutant coexpression was considered evidence to support coassembly of WT-LQT2 mutant heteromeric channels (4, 10, 18, 19, 22). Direct evidence for the physical association of LQT2 mutant and WT subunits was not shown.

The present study was designed 1) to investigate in greater detail the molecular mechanism responsible for the dominant negative effect of N470D mutant subunits coexpressed with WT subunits and 2) to test whether the trafficking defective heteromeric WT-N470D channels could be pharmacologically rescued with a high-affinity HERG channel blocking drug. Our findings show that 1) N470D mutant and WT subunits are physically associated as core-glycosylated protein in the ER to form heteromeric channels that are then retained in the ER to cause decreased cell surface expression of WT HERG subunits and 2) the cell surface expression of heteromeric channels is increased by culturing cells in the presence of the HERG channel blocking drug E-4031. Thus heteromeric channels containing the N470D mutation, as well as homomeric channels, are able to undergo pharmacological rescue.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA constructs and transfections of HEK-293 cells. The HERG mutations and epitope-tagged constructs were generated by site-directed mutagenesis using the Gene-Editor in vitro mutagenesis system (Promega; Madison, WI). For Flag-tagged HERG constructs, the Flag epitope (DYKDDDDK) was inserted in frame at the COOH-terminus of HERG. The constructs were then subcloned into BamHI and EcoRI sites of the pcDNA3 vector (Invitrogen; Carlsbad, CA). For Myc-tagged HERG constructs, the stop codon of HERG was removed and replaced by an EcoRI site. The constructs were then subcloned in-frame into BamHI and EcoRI sites of the pcDNA4-MycA vector (Invitrogen). The hKv1.4 cDNA in the pBK-CMV vector was kindly provided by Dr. Michael Tamkun (26). Cells were transiently or stably transfected with these constructs using a lipofectamine method as described previously (30). For stable cotransfection of Myc-tagged and Flag-tagged constructs, cells were first transfected with Myc-tagged construct and selected by 100 µg/ml zeocin. Cells stably expressing Myc-tagged construct were then transfected with Flag-tagged construct and selected by 400 µg/ml G418. HEK-293 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2.

Western blot and immunoprecipitation. Membrane protein preparation and Western blot procedures were as previously described (30). The membrane proteins were subjected to SDS-PAGE and then electrophoretically transferred onto nitrocellulose membranes. The membranes were incubated with anti-Myc, anti-Flag, or anti-Kv1.4 antibodies (Alomone; Jerusalem, Israel) and visualized with an ECL detection kit.

The immunoprecipitation was performed as described previously (28). Briefly, cells were lysed in immunoprecipitation buffer [containing 10 mM Tris·HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mg/ml BSA, and protease inhibitors]. The cell lysate was immunoprecipitated with anti-Flag antibody. The antigen-antibody complex was subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and detected by Western blot analysis with anti-Myc antibody or anti-Flag antibody. Alternatively, the cell lysate was immunoprecipitated with anti-Myc antibody, followed by Western blot with anti-Flag antibody.

For experiments using endoglycosidase H (Endo H) treatment, the immunoprecipitated proteins were dissolved in 30 µl of 50 mM sodium citrate buffer (pH 5.5) containing 15 mM {beta}-mercaptoethanol and 0.12% SDS by boiling for 2 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 0.5 mM, followed by the addition of 15 milliunits Endo H (Roche; Indianapolis, IN). The mixture was incubated at 37°C for 24 h. Adding sample buffer and boiling for 2 min stopped the reaction.

Immunofluorescence microscopy. The immunofluorescence microscopy was performed as described previously (28). Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and blocked with a buffer containing 5% goat serum, 0.2% Triton X-100, and 0.05% azide in PBS. The cells were then incubated with monoclonal anti-Myc antibody (1:3,000, Convance; Berkley, CA) and polyclonal anti-Flag antibody (1:1,000, Affinity Bioreagents; Golden, CO) at 4°C overnight. After being washed with PBS, the cells were incubated with Alexa 488-conjugated goat anti-mouse IgG secondary antibody and Alexa 594-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR). Immunofluorescence staining was viewed with a Nikon fluorescence microscope.

Patch-clamp recordings. Membrane currents were recorded in the whole cell configuration using suction pipettes as described previously (30). The bath solution contained (in mM) 137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). The pipette solution contained (in mM) 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, and 10 HEPES (pH 7.2 with KOH). An Axopatch-200B patch-clamp amplifier was used to record membrane currents. Computer software (pCLAMP8) was used to acquire and analyze current signals. All patch-clamp experiments were performed at 22–23°C within 1–2 h of removing cells from culture conditions. Data are presented as means ± SE. Student's t-test was used for statistical analysis.

Drug treatment. E4031 (Eisai; Tokyo, Japan) was dissolved in distilled H2O to give a 5 mM stock solution. The final concentration of E-4031 was made by adding the stock solution to the culture medium. For Western blot analysis, cells were cultured in the presence of 5 µM E-4031 for 24 h. For patch-clamp experiments, cells were cultured in the presence of 5 µM E-4031 for 24 h and then cultured in drug-free medium for 1 h to washout the drug.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To demonstrate coassembly of N470D mutant and WT HERG subunits, we used differentially epitope-tagged constructs. We introduced the Myc epitope into WT HERG (WT-Myc) and the Flag epitope into WT HERG (WT-Flag) and N470D (N470D-Flag). These constructs were cotransfected into HEK-293 cells (or transfected alone as a control). Coassembly of WT-Myc with WT-Flag or N470D-Flag was demonstrated by immunoprecipitation with anti-Flag antibody, followed by Western blot analysis with anti-Myc antibody (Fig. 1, top; n = 4). The results show that WT-Myc was coimmunoprecipitated with WT-Flag (lane 4) and N470D-Flag (lane 5). The coimmunoprecipitation was not observed in cells transfected with WT-Myc, WT-Flag, or N470D-Flag alone (lanes 1–3). In some experiments, the immunoprecipitates were also probed with anti-Flag antibody to show the efficient immunoprecipitation of Flag-tagged HERG proteins (Fig. 1, bottom; n = 2). Coassembly of WT-Myc with WT-Flag or N470D-Flag was further confirmed by immunoprecipitation with anti-Myc antibody, followed by Western blot analysis with anti-Flag antibody (data not shown and see Fig. 2). The association of WT-Myc and WT-Flag was observed in both 135- and 155-kDa forms of HERG channel protein. In contrast, the association of WT-Myc and N470D-Flag was observed primarily in the 135-kDa form of HERG channel protein. Because the 135-kDa protein represents the core-glycosylated, immature form of HERG channel protein and the 155-kDa protein represents the complex-glycosylated, mature form of HERG channel protein (6, 28), the present results suggest that coassembly of N470D and WT HERG occurs before the formation of complex glycosylation in HERG channel biosynthesis pathway. To further study the intracellular localization of coassembly of N470D and WT HERG, we performed Endo H digestion experiments. Endo H removes high-mannose oligosaccharides, which are added during core glycosylation of newly synthesized proteins in the ER. Once proteins reach the medial Golgi they undergo complex oligosaccharide modification to become Endo H resistant. In these experiments, WT-Myc and N470D-Flag were coexpressed in HEK-293 cells and subjected to immunoprecipitation with anti-Myc antibody. The coprecipitated channel proteins were then treated with Endo H and analyzed by Western blot with anti-Flag antibody. As shown in Fig. 2, coprecipitated proteins were sensitive to Endo H treatment and showed a decrease in molecular mass from 135 to 132 kDa (n = 2). This result suggests that the coassembly of WT HERG and N470D occurs in a premedium Golgi compartment, probably in the ER.



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  		Fig. 1. Coassembly of Myc-tagged and Flag-tagged human ether-a-go-go-related gene (HERG) channels. HEK-293 cells were stably transfected with wild-type (WT)-Myc (lane 1), WT-Flag (lane 2), N470D-Flag (lane 3), WT-Myc and WT-Flag (lane 4), or WT-Myc and N470D-Flag (lane 5). Cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibody, followed by Western blot (WB) analysis with anti-Myc antibody or anti-Flag antibody as indicated.

 


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  		Fig. 2. Effect of endoglycosidase H (Endo H) on HERG proteins. Cell lysates from cells expressing WT-Myc and N470D-Flag were subjected to immunoprecipitation with anti-Myc antibody. After immunoprecipitation, the samples were treated with (+) or without (–) Endo H and analyzed by Western blot analysis with anti-Flag antibody.

 
Because N470D is a trafficking defective mutant, it is important to determine whether coassembly of the N470D mutant and WT HERG causes WT subunits to also become trafficking defective. Figure 3 shows a representative Western blot (n = 4) of differentially tagged HERG channels. In cells expressing WT-Myc and WT-Flag, Western blot analysis with anti-Myc antibody (recognizes WT-Myc; Fig. 3, lane 1) and anti-Flag antibody (recognizes WT-Flag; Fig. 3, lane 3) showed both the immature and mature forms of HERG channel protein. In contrast, in cells expressing WT-Myc and N470D-Flag, Western blot analysis with anti-Myc antibody (recognizes WT-Myc; Fig. 3, lane 2) and anti-Flag antibody (recognizes N470D-Flag; Fig. 3, lane 4) showed primarily the immature form of HERG channel protein. These results suggest that coexpression of WT HERG and N470D causes a trafficking defect of WT subunits similar to that of N470D subunits. To confirm the biochemical results, we performed immunofluoresence staining of Myc-tagged and Flag-tagged HERG channels (Fig. 4; n = 4–5 cells in each group). In cells expressing WT-Myc and WT-Flag, staining with anti-Myc antibody (Fig. 4B) and anti-Flag antibody (Fig. 4C) showed similar staining patterns throughout the cells, including the surface membrane, suggesting colocalization of Myc-tagged and Flag-tagged WT HERG channel proteins. In cells expressing WT-Myc and N470D-Flag, Myc-tagged WT HERG (Fig. 4E) and Flag-tagged N470D (Fig. 4F) also showed similar staining patterns; however, the staining patterns were more restricted to a perinuclear region. The specificity of anti-Myc and anti-Flag antibodies was tested by staining of cells transfected with WT-Myc or WT-Flag alone, and no cross reactions were observed (data not shown). The immunostaining findings are consistent with our Western blot data and suggest that coassembly of WT HERG and N470D results in a trafficking defect of heteromeric channels. However, it has been reported that expression of misfolded mutant protein may disrupt the ER-to-Golgi trafficking of other unrelated proteins (7). It is possible that defective trafficking of WT HERG is not due to specific coassembly with N470D, but due to nonspecific disruption of ER-to-Golgi trafficking. To rule out this possibility, we studied another human voltage-gated K+ channel, Kv1.4. If expression of N470D causes nonspecific disruption of ER-to-Golgi trafficking, the trafficking of this unrelated potassium channel should also be affected. When expressed in HEK-293 cells, Kv1.4 channel protein exhibited two bands on Western blot analysis: an upper band with a molecular mass of 105 kDa and a lower band with a molecular mass of 85 kDa (data not shown). The lower band represents the immature form of Kv1.4 channel protein located in the ER, and the upper band represents the mature form located in the plasma membrane (14). When Kv1.4 was coexpressed with WT HERG or N470D, it exhibited similar two bands on Western blot, suggesting that the N470D mutant does not disrupt the ER-to-Golgi trafficking of Kv1.4 protein. Therefore, the defective trafficking of WT HERG observed in cells expressing WT-Myc and N470D-Flag is likely caused by coassembly of WT and N470D subunits.



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  		Fig. 3. Western blot analysis of Myc-tagged and Flag-tagged HERG channels coexpressed in HEK-293 cells. Differentially tagged HERG channel proteins were immunoblotted with anti-Myc (lanes 1 and 2) or anti-Flag antibodies (lanes 3 and 4). In cells expressing WT-Myc and WT-Flag, both immature and mature forms of HERG protein are present. In cells expressing WT-Myc and N470D-Flag, only the immature form of HERG protein is present.

 


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  		Fig. 4. Immunofluoresence staining of Myc-tagged and Flag-tagged HERG channels coexpressed in HEK-293 cells. Staining patterns are shown for cells expressing WT-Myc and WT-Flag (A–C) or WT-Myc and N470D-Flag (D–F). For each cell, a phase-contrast photograph (A and D), monoclonal anti-Myc antibody staining (B and E, green images), and polyclonal anti-Flag-antibody staining (C and F, red images) are shown. Calibration bar = 20 µm.

 
We next tested whether trafficking defective HERG channels in cells expressing heteromeric WT-Myc and N470D-Flag channels could be rescued by the HERG channel blocking drug E-4031. In these experiments, cells were cultured at 37°C for 24 h in the absence or presence of 5 µM E-4031, and WT HERG and the N470D mutant were immunoblotted with anti-Myc antibody and anti-Flag antibody, respectively. As shown in Fig. 5A, in the absence of E-4031, WT HERG and N470D heteromeric channels expressed mostly the 135-kDa immature form of HERG proteins, whereas in the presence of E-4031 the 155-kDa mature form of both WT-Myc and N470D-Flag increased (n = 3). In control experiments, Western blot analysis of cells expressing WT-Myc and WT-Flag heteromeric channels showed typical two bands of the 135-kDa immature form and the 155-kDa mature form for both Myc-tagged and Flag-tagged WT HERG channels, and E-4031 had no effect (Fig. 5B; n = 2). To study the effect of E-4031 treatment on the coassembly of WT HERG and the N470D mutant, we performed coimmunoprecipitation experiments in cells expressing WT-Myc and N470D-Flag. As shown in Fig. 5C, WT-Myc was coimmunoprecipitated with N470D-Flag in both control conditions and after cells were cultured in E-4031 (n = 2), suggesting that the presence of E-4031 does not affect the formation of the WT-N470D complex. The result also shows that drug rescue can occur in WT-N470D heteromeric channels.



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  		Fig. 5. Effect of E-4031 on protein trafficking of WT HERG and N470D mutant channels. Cells expressing WT-Myc and N470D-Flag (A and C) or WT-Myc and WT-Flag (B) were cultured in the absence (–) or presence (+) of 5 µM E-4031 for 24 h. Differentially tagged HERG channel proteins were immunoblotted with anti-Myc or anti-Flag antibodies as indicated (A and B). C: cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by Western blot analysis with anti-Myc antibody.

 
The electrophysiological study of pharmacologically rescued channels in cells expressing WT-Myc and N470D-Flag is shown in Fig. 6. In these experiments, HERG current was activated by 4-s-long depolarizing steps between –70 and 50 mV from a holding potential of –80 mV. HERG tail current was recorded after repolarization to –50 mV. When cells were cultured in the absence of E-4031, the HERG current amplitude was small. The maximum peak tail current measured at –50 mV was 8.5 ± 1.2 pA/pF (n = 8 cells; Fig. 6B). When the same cell line was cultured in 5 µM E-4031 for 24 h followed by drug-free medium for 1 h to washout the drug, the HERG current amplitude was increased with a maximum peak tail current of 20.9 ± 1.6 pA/pF (n = 7 cells, P < 0.05; Fig. 6B). We and others have shown that when expressed as homomeric channels, the N470D mutant activates at more negative voltages (~25 mV) than WT HERG channels (22, 29, 30). To study the voltage dependence of HERG channel activation in cells expressing heteromeric WT-Myc and N470D-Flag, we fit the normalized tail currents with a Boltzmann function (Fig. 6C). The half-maximal activation voltages (V1/2) in control conditions and after culture in E-4031 were –16.7 ± 1.5 mV (n = 8 cells) and –28.8 ± 1.6 mV (n = 7 cells), respectively. The slope factors were 7.6 ± 0.4 and 7.6 ± 0.3, respectively. In control conditions, the V1/2 of HERG channels in cells expressing WT-Myc and N470D-Flag was similar to that we previously found for homomeric WT HERG channels (–14.2 mV) (30). This suggests that the small-amplitude current observed in cells expressing WT-Myc and N470D-Flag without culture in E-4031 is generated by channels that are mostly homotetramers formed by WT-Myc subunits. After cells were cultured in E-4031, the V1/2 was shifted from –16.7 to –28.8 mV, a value intermediate between homomeric WT (–14.2 mV) and homomeric N470D (–40.4 mV) channels (29, 30). This is likely to represent the pharmacological rescue of heteromeric channels containing both WT and N470D subunits by E-4031.



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  		Fig. 6. Rescue of HERG current in cells stably expressing WT-Myc and N470D-Flag by E-4031. A: representative currents recorded from cells expressing WT-Myc and N470D-Flag. Cells were cultured in the absence (control; top) or presence of 5 µM E-4031 (bottom) for 24 h. E-4031 was washed out by culturing the cells in drug-free medium for 1 h before recordings. B: current-voltage plot of HERG tail current measured at –50 mV. Triangles indicate control (n = 8 cells), and circles indicate cultured in the presence of E-4031 (n = 7 cells). C: activation curves for control (triangles, n = 8) and after culture in E-4031 (circles, n = 7).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we showed that coexpression of HERG LQT2 N470D subunits with WT subunits results in their physical association in the ER as heteromeric channels. The coassembly results in the retention of the heteromeric WT-N470D channels as trafficking defective proteins, as evidenced by their expression as mostly the core-glycosylated proteins of 135 kDa in Western blot analysis and perinuclear distribution in immunostaining. WT HERG channel protein consists of two bands (135 and 155 kDa) on Western blot analysis, corresponding to the immature and mature forms of HERG channel protein, respectively (28). The generation of these two bands is due to the modification of N-linked glycosylation at residue N598 (6). The 135-kDa band is the core-glycosylated form located in the ER, and the 155-kDa band is the complex-glycosylated, plasma membrane form after processing in the Golgi apparatus. Although N-linked glycosylation is not required for the cell surface expression of functional channels, the presence of the complex glycosylation pattern (155 kDa) on Western blot can be used as a marker for HERG channel trafficking from the ER to the plasma membrane (6). Therefore, our results suggest that the N470D mutant causes dominant negative suppression of WT HERG current by decreasing the cell surface expression of WT HERG channels. Furthermore, we show that the cell surface expression of the heteromeric channels is increased by culturing cells in the presence of the HERG channel blocker E-4031. It has been reported that coexpression of the LQT2 mutation A561V with WT HERG causes ER retention and a decrease in the abundance of WT subunits (4, 10). In contrast to N470D, the trafficking defect of A561V cannot be corrected by the HERG channel blocker E-4031 (4). Two mechanisms may account for how trafficking-defective mutant proteins may affect the normal trafficking of WT proteins. The first mechanism is that mutant and WT subunits form heteromultimeric proteins that exhibit the phenotype of mutant subunits (8, 11, 15). The second mechanism is that misfolded mutant proteins cause nonspecific disruption of protein transport that leads to aberrant trafficking of other proteins destined for the plasma membrane or secretion (7). For HERG channels, the dominant negative effect of the A561V mutation has been proposed to result from the coassembly of mutant and WT subunits as heteromeric channels (4, 10); however, direct evidence for the physical association of LQT2 mutant and WT subunits has not been previously reported. Using immunoprecipitation, followed by Western blot analysis of differentially tagged HERG subunits, we showed direct evidence for the physical association of N470D with WT subunits as the mechanism for the dominant negative effect of the N470D mutant. The ER contains a quality control system that prevents incorrectly assembled or misfolded proteins from exiting the ER. Thus it is likely that misfolded N470D subunits assemble with WT subunits in the ER to cause ER retention of the coassembled channels by the quality control system. Our results also exclude nonspecific disruption of ER to Golgi transport by the N470D mutant as a mechanism for the trafficking defect of WT HERG subunit, because the trafficking of unrelated Kv1.4 channels is not affected by coexpression of Kv1.4 with the N470D subunit. Our findings add to the importance of impaired trafficking of WT proteins caused by mutant proteins as has been shown in other diseases as a mechanism for the dominant negative effect (7, 8, 11, 15).

Furthermore, our findings provide insight as to where the protein trafficking defect occurs in channel biogenesis. The heteromeric WT-Myc and N470D-Flag tagged channels, as shown in Fig. 2, successfully undergo core glycosylation as well as coassembly; thus the channel subunits appear to be processed normally during early translational and posttranslational steps of biogenesis only to then become trafficking defective at subsequent protein processing and sorting steps preceding complex glycosylation.

Defective protein trafficking has been recognized as an important mechanism for an increasing number of inherited human diseases (1). In many cases, trafficking defective mutant proteins are functional if they can be rescued to their final destinations. The concept of chemical and pharmacological chaperones has emerged as a strategy for rescue of trafficking defective proteins, and many different chaperones are now recognized (2, 13, 17, 24). For some LQT2 mutations, one important group are drugs that block HERG channel current, and several of these compounds are known to correct defective trafficking of mutant HERG proteins. We and other investigators have shown that the trafficking defective LQT2 mutations T65P, N470D, and S601G can be rescued by HERG channel blocking drugs (5, 20, 21, 29). HERG channel blocking drugs have no effect on the cell surface expression of WT HERG (5, 29). LQT2 mutations that can be rescued by HERG channel blocking agents appear to express small-amplitude currents under control conditions, and this may be a "signature" for potential pharmacological rescue (21). These LQT2 mutations may represent a mild phenotype with subtle misfolding and a low efficiency of maturation, and HERG channel blocking drugs may act as pharmacological chaperones to increase the maturation efficiency of these mutant channels (5). As shown in Fig. 5, although E-4031 has no effect on the cell surface expression of WT channels, when HERG WT subunits are coexpressed with N470D subunits, E-4031 increased or rescued the mature channel protein (the 155-kDa band) of WT-N470D coassembled channels. Thus pharmacological rescue is not restricted to homomeric mutant channels as we previously reported; rather, the present findings show that pharmacological rescue occurs for heteromeric channels that contain both mutant and WT subunits. This more closely represents the pathophysiological conditions found in LQT2.

The patch-clamp experiments show that electrophysiological properties of the rescued heteromeric channels are altered. In control conditions, where the expression of small numbers of channels containing mostly WT subunits is expected, the V1/2 was –16.7 mV, a value similar to that we previously reported for HERG WT homomeric channels (30). After culture in E-4031, the 155-kDa protein band and HERG current density were increased; however, the V1/2 was shifted negatively to –28.8 mV. The likely explanation is that pharmacological rescue with E-4031 increased the density of WT-N470D heteromeric channels (plus a smaller number of N470D homomeric channels) in the plasma membrane by reducing the dominant negative protein trafficking effect, yet the increased presence of N470D subunits resulted in the negative shift in the V1/2 to a value intermediate between homomeric WT and homomeric N470D channels. Thus, although the pharmacological rescue of trafficking defective mutant-containing HERG channels may increase the delivery of functional protein to the surface membrane, the electrophysiological phenotype of the rescued channels is affected by the biophysical properties of N470D mutant subunits.


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 ABSTRACT
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This study was supported in part by a Medical Research Foundation of Oregon grant (to Z. Zhou), National Heart, Lung, and Blood Institute Grants HL-68854 (to Z. Zhou) and HL-60723 (to C. T. January), and an award to Oregon Health and Science University under the Howard Hughes Medical Institute Biomedical Research Support Program for Medical Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Zhou, Div. of Molecular Medicine, NRC3, Oregon Health and Science Univ., 3181 SW Sam Jackson Park Rd., Portland, OR 97239 (E-mail: zhouzh{at}ohsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Aridor M and Hannan LA. Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 1: 836–851, 2000.[CrossRef][ISI][Medline]
  2. Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, and Welch WJ. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1: 117–125, 1996.[CrossRef][ISI][Medline]
  3. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, and Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80: 795–803, 1995.[CrossRef][ISI][Medline]
  4. Ficker E, Dennis AT, Obejero-Paz CA, Castaldo P, Taglialatela M, and Brown AM. Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome. J Mol Cell Cardiol 32: 2327–2337, 2000.[CrossRef][ISI][Medline]
  5. Ficker E, Obejero-Paz CA, Zhao S, and Brown AM. The binding site for channel blockers that rescue misprocessed human long QT syndrome type 2 ether-a-go-go-related gene (HERG) mutations. J Biol Chem 277: 4989–4998, 2002.[Abstract/Free Full Text]
  6. Gong Q, Anderson CL, January CT, and Zhou Z. Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283: H77–H84, 2002.[Abstract/Free Full Text]
  7. Graves TK, Patel S, Dannies PS, and Hinkle PM. Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic. J Cell Sci 114: 3685–3694, 2001.[ISI][Medline]
  8. Ito M, Yu RN, Jameson JL, and Ito M. Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes insipidus retain dimerization and impair the secretion of wild-type proteins. J Biol Chem 274: 9029–9037, 1999.[Abstract/Free Full Text]
  9. January CT, Gong Q, and Zhou Z. Long QT syndrome: cellular basis and arrhythmia mechanisms in LQT2. J Cardiovasc Electrophysiol 11: 1413–1418, 2000.[CrossRef][ISI][Medline]
  10. Kagan A, Yu Z, Fishman GI, and McDonald TV. The dominant negative LQT2 mutation A561V reduces wild-type HERG expression. J Biol Chem 275: 11241–1248, 2000.[Abstract/Free Full Text]
  11. Kamsteeg EJ, Wormhoudt TA, Rijss JP, van Os CH, and Deen PM. An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus. EMBO J 18: 2394–2400, 1999.[CrossRef][ISI][Medline]
  12. Keating MT and Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104: 569–580, 2001.[CrossRef][ISI][Medline]
  13. Loo TW and Clarke DM. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. J Biol Chem 272: 709–712, 1997.[Abstract/Free Full Text]
  14. Manganas LN, Wang Q, Scannevin RH, Antonucci DE, Rhodes KJ, and Trimmer JS. Identification of a trafficking determinant localized to the Kv1 potassium channel pore. Proc Natl Acad Sci USA 98: 14055–14059, 2001.[Abstract/Free Full Text]
  15. Marr N, Bichet DG, Lonergan M, Arthus MF, Jeck N, Seyberth HW, Rosenthal W, van Os CH, Oksche A, and Deen PM. Heteroligomerization of an Aquaporin-2 mutant with wild-type Aquaporin-2 and their misrouting to late endosomes/lysosomes explains dominant nephrogenic diabetes insipidus. Hum Mol Genet 11: 779–789, 2002.[Abstract/Free Full Text]
  16. Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, and Bennett V. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421: 634–639, 2003.[CrossRef][Medline]
  17. Morello JP, Salahpour A, Laperriere A, Bernier V, Arthus MF, Lonergan M, Petaja-Repo U, Angers S, Morin D, Bichet DG, and Bouvier M. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest 105: 887–895, 2000.[ISI][Medline]
  18. Nakajima T, Furukawa T, Tanaka T, Katayama Y, Nagai R, Nakamura Y, and Hiraoka M. Novel mechanism of HERG current suppression in LQT2: shift in voltage dependence of HERG inactivation. Circ Res 83: 415–422, 1998.[Abstract/Free Full Text]
  19. Nakajima T, Kurabayashi M, Ohyama Y, Kaneko Y, Furukawa T, Itoh T, Taniguchi Y, Tanaka T, Nakamura Y, Hiraoka M, and Nagai R. Characterization of S818L mutation in HERG C-terminus in LQT2. Modification of activation-deactivation gating properties. FEBS Lett 481: 197–203, 2000.[CrossRef][ISI][Medline]
  20. Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, and Aerssens J. A novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency. J Biol Chem 277: 48610–48616, 2002.[Abstract/Free Full Text]
  21. Rajamani S, Anderson CL, Anson BD, and January CT. Pharmacological rescue of human K+ channel long-QT2 mutations: human ether-a-go-go-related gene rescue without block. Circulation 105: 2830–2835, 2002.[Abstract/Free Full Text]
  22. Sanguinetti MC, Curran ME, Spector PS, and Keating MT. Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci USA 93: 2208–2212, 1996.[Abstract/Free Full Text]
  23. Sanguinetti MC, Jiang C, Curran ME, and Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81: 299–307, 1995.[CrossRef][ISI][Medline]
  24. Sato S, Ward CL, Krouse ME, Wine JJ, and Kopito RR. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J Biol Chem 271: 635–638, 1996.[Abstract/Free Full Text]
  25. Splawski I, Shen J, Timothy KW, Lehmann MH, Priori S, Robinson JL, Moss AJ, Schwartz PJ, Towbin JA, Vincent GM, and Keating MT. Spectrum of mutations in long-QT syndrome genes KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation 102: 1178–1185, 2000.[Abstract/Free Full Text]
  26. Tamkun MM, Knoth KM, Walbridge JA, Kroemer H, Roden DM, and Glover DM. Molecular cloning and characterization of two voltage-gated K+ channel cDNAs from human ventricle. FASEB J 5: 331–337, 1991.[Abstract]
  27. Trudeau MC, Warmke JW, Ganetzky B, and Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92–95, 1995.[Abstract/Free Full Text]
  28. Zhou Z, Gong Q, Epstein ML, and January CT. HERG channel dysfunction in human long QT syndrome: intracellular transport and functional defects. J Biol Chem 273: 21061–21066, 1998.[Abstract/Free Full Text]
  29. Zhou Z, Gong Q, and January CT. Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome: pharmacological and temperature effects. J Biol Chem 274: 31123–31126, 1999.[Abstract/Free Full Text]
  30. Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, and January CT. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J 74: 230–241, 1998.[Medline]



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