Kv4.3 Modulates the Distribution of hERG.
Journal: 2018/November - Scientific Reports
ISSN: 2045-2322
Abstract:
This study examines the interaction between hERG and Kv4.3. The functional interaction between hERG and Kv4.3, expressed in a heterologous cell line, was studied using patch clamp techniques, western blot, immunofluorescence, and co-immunoprecipitation. Co-expression of Kv4.3 with hERG increased hERG current density (tail current after a step to +10 mV: 26 ± 3 versus 56 ± 7 pA/pF, p < 0.01). Kv4.3 co-expression also increased the protein expression and promoted the membrane localization of hERG. Western blot showed Kv4.3 increased hERG expression by Hsp70. hERG and Kv4.3 co-localized and co-immunoprecipitated in cultured 293 T cells, indicating physical interactions between hERG and Kv4.3 proteins in vitro. In addition, Hsp70 interacted with hERG and Kv4.3 respectively, and formed complexes with hERG and Kv4.3. The α subunit of Ito Kv4.3 can interact with and modify the localization of the α subunit of IKr hERG, thus providing potentially novel insights into the molecular mechanism of the malignant ventricular arrhythmia in heart failure.
Relations:
Content
Similar articles
Articles by the same authors
Discussion board
Scientific Reports. Dec/31/2016; 7
Published online Dec/18/2017

Kv4.3 Modulates the Distribution of hERG

Abstract

This study examines the interaction between hERG and Kv4.3. The functional interaction between hERG and Kv4.3, expressed in a heterologous cell line, was studied using patch clamp techniques, western blot, immunofluorescence, and co-immunoprecipitation. Co-expression of Kv4.3 with hERG increased hERG current density (tail current after a step to +10 mV: 26 ± 3 versus 56 ± 7 pA/pF, p < 0.01). Kv4.3 co-expression also increased the protein expression and promoted the membrane localization of hERG. Western blot showed Kv4.3 increased hERG expression by Hsp70. hERG and Kv4.3 co-localized and co-immunoprecipitated in cultured 293 T cells, indicating physical interactions between hERG and Kv4.3 proteins in vitro. In addition, Hsp70 interacted with hERG and Kv4.3 respectively, and formed complexes with hERG and Kv4.3. The α subunit of Ito Kv4.3 can interact with and modify the localization of the α subunit of IKr hERG, thus providing potentially novel insights into the molecular mechanism of the malignant ventricular arrhythmia in heart failure.

Introduction

Cardiac repolarization is driven by potassium (K+) currents flowing through K+ channels. More specifically, the transient outward potassium current (Ito) is responsible for the early repolarization (phase 1) of the cardiac action potential1,2, while the rapid (IKr) and slow (IKs) delayed rectifier potassium currents are responsible for the rapid repolarization phase (phase 3)3,4. First proposed by Roden in 19985, “repolarization reserve” describes that the inhibition of a current, for example IKr, will not lead to failure of repolarization. Instead, another current, IKs, which remains activated, will act and prevent excessive repolarization delays68. This compensatory potential is lost when IKs is inhibited simultaneously, thus enhancing the effects of IKr inhibition and increasing the consequent risk of excessive repolarization delay and arrhythmias6,7,9. The “repolarization reserve” was then revised10 to that the loss of a single K+ current will not completely impair repolarization (and cause, for example, marked QT prolongation) because of redundancy of K+ currents; when one K+ current is dysfunctional, the other K+ current will increase to compensate so that the changes in action potential duration (APD) are minimized. This compensation requires delicate interaction between the different K+ currents.

A classic voltage-gated K+ channel (Kv channel) consists of four pore-forming (α) subunits that contain the voltage sensor and ion selectivity filter and accessory regulating (β) subunits11,12. For example, the co-assembly of α (KvLQT1) with β (minK) subunits forms IKs channels, while the co-assembly of α subunits encoded by the human ether a-go-go-related gene (hERG)13 and β subunits encoded by the KCNE2 gene product minK-related peptide 1 (MiRP1)14,15 are responsible for the formation of IKr, although the contribution of MiRP1 to native IKr remains uncertain. There is a consensus that the pore-forming α subunits of the Kv4 subfamily, Kv4.3 (KCND3), encodes Ito channels16. However, β subunits have also been proposed to be part of myocardial Ito channels1719. Delpon et al. suggested that the co-association of β subunits of MiRP1 with α subunits of the Kv4.3 contribute to the function of Ito channels19.

Studies show that different K+ currents interacted with each other through α-α subunits or α-β subunits. It is well established that the interaction between IKr and IKs is patho-physiologically relevant20. The prolongation of APD because of a reduction in IKr favors the activation of IKs, which prevents an excessive repolarization delay, and thus suggests potential interactions between the α subunits of IKr and IKs. In 2004, Ehrlich et al. demonstrated that the α subunit of IKs KvLQT1 could interact with the α subunit of IKr hERG to enhance hERG current densities and increase hERG-protein membrane localization20. Wu et al. also suggested that the α subunits of Ito Kv4.3 interact with the β subunits of IKr MiRP1, forming α-β subunits interaction, which is critical for the normal function of the native Ito channel complex in a human heart21.

During heart failure, electrophysiological and structural remodeling changes occur22,23. hERG and Kv4.3 mRNAs, and protein expression levels decrease24. IKr current and Ito current densities are reduced25,26. However, it has not been reported if the α subunit of IKr hERG interacts with the α subunit of Ito Kv4.3 during heart failure. Therefore, in light of the “repolarization reserve” theory, our present study aim to investigate the interactions between the α subunits of IKr and Ito using heterologous cell line co-transfected with hERG and Kv4.3.

Results

Effects of Kv4.3 on hERG current

Figure 1A shows currents recorded from cells expressing hERG without and with Kv4.3 co-transfection. Incubating the cells with 5 nM dofetilide significantly reduced the recorded current, confirming that the currents recorded are hERG currents (IhERG, Fig. 1B). Co-transfection of Kv4.3 increased IhERG densities, compared with cells transfected with hERG alone. Step (Fig. 1C) and tail (Fig. 1D) IhERG densities were significantly larger in co-transfected cells (tail current after a step to +10 mV: 26 ± 3 versus 56 ± 7 pA/pF, p < 0.01)

Figure 1

Co-transfection of Kv4.3 and hERG increases the hERG currents. A and B, recordings from 293 T cells transfected with hERG without (A) or with (B) Kv4.3 co-transfection (voltage protocol in the inset). Co-transfection of Kv4.3 and hERG resulted in significantly greater hERG tail currents (C) and step currents (D) than those transfected with hERG alone. The steady-state activation (E) and inactivation (F) in the cells transfected with hERG alone did not differ from cells co-transfected with Kv4.3. The activation time constants (G), the deactivation time constants (H) and the time constant of recovery (I) were unaltered by co-expression of Kv4.3. *p < 0.05; **p < 0.01; n = 15 and 20 for hERG and hERG + Kv4.3, respectively.

There were no differences observed in the voltage dependence of activation of cells expressing hERG alone or co-expressed with Kv4.3 (V1/2 −13.89 ± 3.77675 versus −13.26 ± 1.27843 mV, n = 15 and 20, respectively, p = NS), (Fig. 1E), and in the steady-state inactivation voltage-dependence (V1/2 −93.57 ± 4.3758 for hERG versus −93.27 ± 4.0758 mV for hERG ±Kv4.3, n = 20, p = NS), (Fig. 1F). The activation time constants were unaltered by co-expression of Kv4.3 (Fig. 1G). However, the deactivation time constants upon repolarization to −50 mV after a 1.5 s step to +20 mV were smaller in cells co-transfected with hERG +Kv4.3 (4.25 ± 0.451 s, n = 18) versus those with hERG alone (3.33 ± 0.250 s, n = 12), (Fig. 1H). The time constant of recovery of cells expressing

hERG alone showed no significant difference when compared with cells co-expressed with hERG and Kv4.3 (Fig. 1I).

Effects of hERG on Kv4.3 current

Since the expression of Kv4.3 affected IhERG, we next determine whether the reversed could also be true; i.e., whether expression of hERG alters Ito currents. Figure 2A shows representative recordings of currents from the cells transfected with Kv4.3 alone or both Kv4.3 and hERG. We also confirmed the currents recorded is Ito because the currents were significantly reduced by 5 nM 4-AP (Fig. 2A, the right panel). Co-transfection of hERG did not affect Kv4.3 current densities; there was no significant difference between those two groups (Fig. 2B). No differences in steady-state activation voltage-dependence were observed (V1/2–20.86 ± 3.10 for Kv4.3 versus −19.82 ± 4.18 mV for Kv4.3 + hERG, p = NS), (Fig. 2C). Similarly, steady-state inactivation voltage-dependence was unaffected (V1/2–80.86 ± 4.98 for Kv4.3 versus −79.82 ± 5.08 mV for Kv4.3 + hERG, p = NS), (Fig. 2D).

Figure 2

Co-transfection of Kv4.3 and hERG has no effect on the Kv4.3 currents. (A) Recordings from 293 T cells transfected with Kv4.3 with or without hERG co-transfection (voltage protocol in the inset). (B) The currents were the same in cells transfected with Kv4.3 or in cells co-transfected with hERG. No differences were observed in the steady-state activation (C) and inactivation (D) of cells transfected with Kv4.3 and Kv4.3 + hERG.

Effects of Kv4.3 on hERG protein expression and localization

The western blot showed that co-transfection of Kv4.3 increased the protein levels of the fully glycosylated mature hERG (155 kDa), but not the core-glycosylated, immature form (135 kDa). However, the protein expression of Kv4.3 was not altered by the co-transfection of hERG (Fig. 3A and C). Confocal imaging of cells transfected with only hERG displayed a strong hERG fluorescence in the cytoplasm; while cells co-transfected with hERG and Kv4.3 showed a homogeneous pattern of increased fluorescence throughout the cytoplasm and cell membrane. However, the distribution or density of Kv4.3 was not altered in cells co-transfected with Kv4.3 and hERG when compared with cell transfected with Kv4.3 only (Fig. 3B).

Figure 3

Co-transfection of Kv4.3 and hERG enhances the protein expression of hERG. The upper panel (A) shows the co-transfection of Kv4.3 increased the protein level of the fully glycosylated mature hERG (155 kDa). However, the lower panel (A) shows that the co-transfection of hERG did not increase the protein expression of Kv4.3. (B) Confocal microscope image of a cell transiently transfected with hERG or Kv4.3 alone (left) or co-transfected with hERG and Kv4.3 (right). The upper panel (B) Red represents immunofluorescence of secondary antibody to anti-hERG. Greater fluorescence was observed (right) at the whole cell compared with the left. The lower panel (B) Green represents Kv4.3 fluorescence signal (FITC-coupled secondary antibody). (C) Quantitative analysis of the data in (A).

Involvement of Hsp70 in Kv4.3-induced hERG expressions

In our experiment, an increase in Hsp70 was observed in cells transfected with both hERG and Kv4.3 (Fig. 4A). Since previous study showed that overexpression of Hsp70 increased the expression of hERG protein27, we therefore hypothesized that Kv4.3 increased the hERG protein expression via upregulation of Hsp70. To test the hypothesis, we examined the expression of hERG in cells transfected with both hERG and Kv4.3, in the presence or absence of Hsp70 siRNA or VER155008, a novel adenosine-derived inhibitor of Hsp7028,29. The results showed that downregulation of Hsp70 using siRNA (Fig. 4B & C) or VER155008 (Fig. 4D and E) effectively abolished the elevation of matured hERG protein expression in the cells co-transfected with hERG and Kv4.3. In addition, as shown in Fig. 4F, absence or presence of hERG transfection did not affect the expression of Hsp70 induced by Kv4.3 transfection, further indicating that hERG itself has no role in the Hsp70 expression. In summary, these results suggest that Kv4.3 enhances hERG protein expression through Hsp70.

Figure 4

Hsp70 mediates the Kv4.3-enhanced hERG protein expression. (A) Co-transfection of Kv4.3 and hERG increased Hsp70 protein expression. (B) Cells co-transfected with hERG and Kv4.3 were preincubated with a dose of (10, 20, 50 and 100 μM) Hsp70 siRNA for 48 h. (C) The hERG protein expression levels in cells co-transfected with Kv4.3 and hERG preincubated with 50 μM Hsp70 siRNA were reduced to the same level as in cells transfected with hERG alone. (D) Cells co-transfected with hERG and Kv4.3 preincubated with a dose of (3, 5, 8 and 10 nM) VER155008 for 24 h. (E) The hERG protein expression levels in cells co-transfected with Kv4.3 and hERG preincubated with 3 nM VER155008 were reduced to the same level as in cells transfected with hERG alone. (F) Absence or presence of hERG transfection did not affect the expression of Hsp70 induced by Kv4.3.

Interactions between hERG and Kv4.3

Double immunofluorescent staining using anti-hERG and anti-Kv4.3 antibody revealed that hERG and Kv4.3 co-localize predominantly at the membrane (Fig. 5A). To assess the possible physical interaction between the two α subunits, immunoprecipitation experiments were carried out in 293 T cells transiently transfected with either hERG or Kv4.3, or co-transfected with hERG and Kv4.3. The whole-cell lysates from cells co-transfected with hERG and Kv4.3 served as an input control. Figure 5B shows that Kv4.3 was precipitated by anti-hERG antibody from the whole-cell lysate of cells co-transfected with hERG and Kv4.3. hERG proteins, both the 135 kDa and 155 kDa forms, were also detected in a reciprocal IP with the Kv4.3 antibody (Fig. 5C). These data showed that hERG co-localize and bound with Kv4.3.

Figure 5

Interaction and complex formation between hERG and Kv4.3. (A) Red represents the immunofluorescence of secondary antibody to anti-hERG. Green represents the Kv4.3 signal. Yellow staining indicates co-localization of hERG and Kv4.3. Proteins were immunoprecipitated with (B) anti-hERG or (C) anti-Kv4.3 and immunoblotted with anti-hERG and anti-Kv4.3. Whole-cell lysates from cells co-transfected with Kv4.3 and hERG served as input control. The co-immunoprecipitation shows that hERG and Kv4.3 indeed interacted with each other.

Interactions of Hsp70 with hERG and Kv4.3

Because Hsp70 was reported to interact with hERG27 and the present study showed that hERG interacts with Kv4.3, we hypothesize that Hsp70 may form complexes with hERG and Kv4.3. We therefore perform immunoprecipitation using an anti-hERG antibody and found that, Hsp70 was precipitated from the whole-cell lysate of cells transfected with hERG and cells co-transfected with hERG and Kv4.3 (Fig. 6A). Further, Hsp70 was precipitated, with an anti-Kv4.3 antibody, from the whole-cell lysate of cells transfected with Kv4.3 and co-transfected with hERG and Kv4.3 (Fig. 6B). Of note, hERG and Kv4.3 were precipitated, with an anti-Hsp70 antibody, from the whole-cell lysate of all groups (Fig. 6C). Interestingly, down-regulation of Hsp70 using siRNA did not affect any of the immunoprecipitation results mentioned above. These data indicate that Hsp70 forms a complex with hERG and Kv4.3; however, absence of Hsp70 does not prevent the complex formation between hERG and Kv4.3.

Figure 6

Hsp70 interacts and forms complexes with hERG and Kv4.3. Inhibiting Hsp70 protein expression did not affect the interaction between hERG and Kv4.3. Proteins were immunoprecipitated with (A) anti-hERG, (B) anti-Kv4.3, or (C) anti-Hsp70. (D) Whole-cell lysates from cells transfected with hERG or Kv4.3 alone, co-transfected with hERG /Kv4.3 or co-transfected with hERG/Kv4.3 and Hsp70 siRNA served as input control.

Interactions of Hsp90 with hERG and Kv4.3

Hsp90 was also reported to enhance hERG protein expression30. However, our results showed that the protein expression of Hsp90 were not affected by hERG or hERG/Kv4.3 transfection (Fig. 7D). Interestingly, Hsp90 protein expression was precipitated, with an anti-hERG antibody, from the whole-cell lysate of hERG and Kv4.3 co-transfected cells, but not from the cells transfected with hERG alone (Fig. 7A). Conversely, Hsp90 protein expression was precipitated, with an anti-Kv4.3 antibody, from whole-cell lysate of cells transfected with Kv4.3 alone, but not from cells co-transfected with hERG and Kv4.3 (Fig. 7B). Immunoprecipitation with anti-Hsp90 showed that Hsp90 interacted with hERG in cells co-transfected with hERG and Kv4.3. However, Hsp90 only interacted with 135 kDa hERG and not with 155 kDa hERG (Fig. 7C).

Figure 7

Hsp90 enhances hERG protein expression in cells co-transfected with hERG and Kv4.3. Proteins were immunoprecipitated with (A) anti-hERG, (B) anti-Kv4.3, or (C) anti-Hsp90. (D) Whole-cell lysates from cells transfected with hERG or Kv4.3 alone, or co-transfected with hERG/Kv4.3 served as input control. Co-immunoprecipitation showed that Hsp90 did not interact with hERG alone; however, Hsp90 interacted with the 135KD immature hERG in cells co-transfected with hERG and Kv4.3. Hsp90 interacted with Kv4.3 in cells transfected with Kv4.3 alone, but not in cells co-transfected with hERG and Kv4.3.

Discussion

In the present study, we have demonstrated physical interactions between Kv4.3 and hERG that were associated with increased hERG-protein expression and enhanced current density without altering the biophysical properties of hERG currents. On the other hand, co-expression with hERG did not alter the cellular distribution of Kv4.3 or the density or property of Kv4.3 currents.

hERG encodes the α subunit of IKr. Kv4.3 is the α subunit of human Ito. Our study indicates a novel potential molecular interaction between these two α subunits of hERG and Kv4.3. Kv4.3-induced hERG protein expression were paralleled by an increase in IhERG density. In the presence of Kv4.3, IhERG deactivation time constants were smaller, but no difference was found in the steady-state activation and inactivation. Also, the co-transfection of hERG showed no effect on the protein expression and the biophysical properties of Kv4.3.

Under physiological conditions, the wild type hERG exhibits two bands on Western blot analysis; this represents different stages of asparagine (Asn)-linked glycosylation31. The core-glycosylated, immature form is represented by a 135 kDa band and is present in the endoplasmic reticulum (ER); the fully glycosylated mature protein is observable as a 155 kDa band and represents hERG localizes either in the Golgi apparatus or on the cell surface32,33. The comparison of the intensity of these two bands provides a useful details regarding hERG trafficking. We found that the mature form of hERG (155 kDa) increased after the cells were co-transfected with Kv4.3. However, since transient transfection was used in our experiments, majority of the protein may still be within intracellular milieu (immature). These results were further verified by confocal imaging. In the presence of Kv4.3, more hERG protein localized on the surface of the cells, thus suggesting that Kv4.3 enhances hERG trafficking.

Ficker et al. showed that the cytosolic chaperones Hsp70 interacted with hERG during maturation27. The Hsp70 family, including stress-induced Hsp70 and constitutively expressed heat shock cognate protein (Hsc)70, was shown to interact with the core-glycosylated form of hERG27,34 and increases the levels of mature form of hERG27. Based on aforementioned studies, we hypothesize that Hsp70 may be involved in the maturation of hERG induced by Kv4.3. Indeed, we found that Hsp70 protein expressions were increased in cells co-transfected with hERG and K4.3. The upregulation of hERG expression by Kv4.3 is dependent on Hsp70, as Hsp70 siRNA could prevent the Kv4.3-induced hERG expression. Similar result was obtained when Hsp70 inhibitor, VER155008, was used. These results suggest that Kv4.3 enhances hERG protein trafficking through Hsp70.

Since the confocal image demonstrated that hERG and Kv4.3 co-localized on cell surface, we further investigated the interaction between hERG and Kv4.3 using immunoprecipitation. The result showed that hERG and Kv4.3 bind to each other and form a complex. Both mature and non-mature forms of hERG co-immunoprecipitated with Kv4.3.

Interactions between hERG and potential α and β subunits have been suggested. For instances, KvLQT1 was shown to interact with hERG to modulate hERG localization and current densities20. KvLQT1 could also interact with hERG at the C-terminal cytoplasmic tails of hERG that contained cyclic nucleotide binding domain20. MiRP1 is also believed to interact with hERG at the C-terminal region35,36. It is known that long-QT syndrome type 2 (LQT2) mutations in the C-terminal tail affect trafficking of hERG from the ER to the cell surface3739. A number of the C-terminal interacting proteins such as GM130 and 14–3–3 have been identified in playing potential roles in the trafficking of hERG to the cell surface4042. However, in our study, we did not determine which domain of hERG interacted with Kv4.3.

We observed that Kv4.3 interacted with hERG and increased hERG expression through Hsp70. It has been reported that hERG formed complexes with Hsp7027. Therefore, we tested if Hsp70 formed complexes with hERG and Kv4.3, and if Hsp70 mediated the interactions between hERG and Kv4.3. Our data demonstrated that both Kv4.3 and hERG interacted with Hsp70, suggesting that Hsp70 may form complexes with hERG and Kv4.3. However, although downregulation of Hsp70 decreased hERG protein expression in cells co-transfected with Kv4.3 and hERG, Hsp70 did not play a critical role in the complex formation between hERG and Kv4.3. These data indicate that Hsp70 increases hERG protein expression and forms complexes with hERG via two independent mechanisms.

Hsp90 only interacted with Kv4.3, and not with hERG when expressed alone (Fig. 7). However, Hsp90 preferably interacted with hERG in cells co-transfected with hERG and Kv4.3 and enhanced the expression of 135 kDa hERG. These results suggest that Hsp90 plays a very complicated role in the intereaction between hERG and Kv4.3, which warrants further study.

There are a few limitations in the present study. In all experiments, hERG and Kv4.3 were transiently transfected in cells; therefore, the immature form of hERG had higher band intensity than the mature form. Our studies were performed primarily in HEK293T cells. It remained to be determined whether the interactions that we observed were specific to this cell line. The association between hERG and Kv4.3 was not verified in cardiac myocytes or animal model. Therefore caution should be taken to extrapolate the findings to cardiac myocytes because cardiac myocytes express many other ion channels which may increase or decrease the interaction between hERG and Kv4.3.

Conclusion

In conclusion, our finding reveals the interaction between the α subunits of IKr hERG and Ito Kv4.3. Kv4.3 modulates hERG current density by promoting hERG protein expression and localization via Hsp70. Kv4.3 and hERG co-localize with each other, and form complexes with Hsp70 in HEK293T cells. The physical and functional interactions between the hERG and Kv4.3 enrich the theory of “repolarization reserve”.

Materials and Methods

Cell culture and transfection

hERG cDNA and Kv4.3 cDNA were provided by Dr. Silvia G. Priori (University of Pavia, Pavia, Italy). Human embryonic kidney 293 (HEK293T) were cultured at 37 °C with 5% CO2 in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). HEK293T cells were transfected transiently or stably with these constructs using a lipofectamine method as described previously20. After the transient transfection, HEK293T cells were studied at 48 hours. When the transiently transfected cells were used for patch clamp experiments, CD8 cDNA was co-transfected to be used as a reporter gene (Invitrogen). CD8 (1 µg) was co-transfected with hERG cDNA (1 µg) or Kv4.3 cDNA (1 µg). CD8-positive cells were identified using Dynabeads (Dynal, M-450 CD8). Cells were harvested between 48–72 hours after the transfection.

Patch Clamp Experiments

The HEK293T cells were bathed in the solution containing (in mmol/L) NaCl 140, KCl 4, CaCl2 2, MgCl2 1, HEPES 10 and glucose 5, adjusted to pH 7.4 with NaOH. The current was recorded by the whole cell patch-clamp technique using a MultiClamp 700B amplifier (Axon Instruments, USA). All signals were acquired at 5 kHz (Digidata 1322 A, Axon Instruments, USA). Patch pipettes were pulled from borosilicate glass on a P-97 horizontal puller (Sutter Instruments, USA). The electrodes had a resistance of 2–3 MΩ. In order to record the K+ currents, pipettes were filled with (in mmol/L): K-aspartame acid 140, MgATP 4, MgCl2 1, EGTA 10, GTP 0.1, HEPES 10 and adjusted to pH 7.3 with KOH.

Ito was elicited by a 300 ms depolarizing pulse from a holding potential of −80 mV to a testing potential of +70 mV and a conditioning test of −40 mV for 25 ms to eliminate the sodium current. This current was blocked by 5 nM 4-aminopyridine (4-AP). IhERG was investigated by applying voltage commands of 5000 ms duration from a holding potential of −80 mV to a range of test potentials from −70 mV to +50 mV, followed by a repolarizing step to −40 mV for 3000 ms to elicit tail currents. This current was significantly inhibited after exposure to 5 nM dofetilide. Average I/V relationships were used to compare current densities following the normalization of current traces to individual cell capacitance measurements. Steady-state I/V relationships were measured at the end of the test pulse.

Immunofluorescence and Confocal Microscopy

For immunofluorescent studies, transiently transfected HEK293T cells were grown on glass bottom cell culture dishes for 24 hours. Cells were then fixed (10 min) with 5% paraformaldehyde (Sigma), washed 3 times (5 min) with a phosphate-buffered saline, blocked with a 3% bovine serum albumin (Sigma), and then permeabilized with 0.1% Triton X-100 (Sigma) for 10 minutes. Cells were incubated overnight (4 °C) with primary antibodies (anti-hERG Alomone, rabbits,) at 1:800 dilution followed by three 5-minutes washes with phosphate-buffered saline. Next, cells were incubated with Alexa Fluor 555 donkey anti-rabbit secondary antibody (Invitrogen) for 45 minutes at room temperature. After being washed, the cells were treated with 4′6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich) for 5 minutes and analyzed under a confocal microscope.

Western Blot Analysis and Co-immunoprecipitation (co-IP)

Whole cell proteins from HEK293T cells that expressed various channels were used for analysis. Proteins were separated on 8% or 12% SDS-polyacrylamide electrophoresis gels, transferred onto nitrocellulose membrane, and blocked for 1 hour with 5% nonfat milk. The blots were incubated with the primary antibody for 24 hours at 4 °C and then incubated with a horseradish peroxidase-conjugated secondary antibody. GAPDH expression was used as loading controls. The blots were visualized with Fujifilm using the ECL detection kit (GE Healthcare). For immunoprecipitation, a whole cell protein (0.5 mg) or cell surface protein was incubated with the appropriate primary antibody overnight at 4 °C and then precipitated with protein A/G plus agarose beads (Santa Cruz) for 4 hours at 4 °C. The beads were washed three times with an ice-cold radio immune precipitation assay lysis buffer, resuspended in 2 mL sample buffer, and boiled for 5 minutes. The samples were centrifuged at 20,000 g for 5 minutes, and the supernatants were collected and analyzed using Western blot.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Footnotes

Xiao-Jing Zhao and Chao Zhu contributed equally to this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [81170177].

Author Contributions

X.-J.Z., Y.H., and Y.L. conceived and designed the experiments. X.-J.Z. and C.Z. executed the experiments. L.-Y.T. analyzed the data. Y.-C.F. and Y.Z. interpreted the data. X.-J.Z. wrote the manuscript. X.-J.Z., C.Z., L.-Y.T., Y.-F.C., Y.Z., X.C., Y.H., and Y.L. critically reviewed the manuscript.

Competing Interests

The authors declare that they have no competing interests.

References

  • 1. GreensteinJLWuRPoSTomaselliGFWinslowRLRole of the calcium-independent transient outward current I(to1) in shaping action potential morphology and durationCirc Res20008710261033[PubMed][Google Scholar]
  • 2. NiwaNNerbonneJMMolecular determinants of cardiac transient outward potassium current (I(to)) expression and regulationJ Mol Cell Cardiol2010481225[PubMed][Google Scholar]
  • 3. SanguinettiMCJurkiewiczNKTwo components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol199096195215[PubMed][Google Scholar]
  • 4. BalserJRBennettPBRodenDMTime-dependent outward current in guinea pig ventricular myocytesGating kinetics of the delayed rectifier. J Gen Physiol199096835863[PubMed][Google Scholar]
  • 5. RodenDMTaking the “idio” out of “idiosyncratic”: predicting torsades de pointesPacing Clin Electrophysiol19982110291034[PubMed][Google Scholar]
  • 6. VarroAThe role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarizationJ Physiol2000523Pt 16781[PubMed][Google Scholar]
  • 7. BiliczkiPViragLIostNPappJGVarroAInteraction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserveBr J Pharmacol2002137361368[PubMed][Google Scholar]
  • 8. RudyYModelling the molecular basis of cardiac repolarizationEuropace20079Suppl 6vi1719[PubMed][Google Scholar]
  • 9. ZengJLauritaKRRosenbaumDSRudyYTwo components of the delayed rectifier K + current in ventricular myocytes of the guinea pig type. Theoretical formulation and their role in repolarizationCirc Res199577140152[PubMed][Google Scholar]
  • 10. RodenDMRepolarization reserve: a moving targetCirculation2008118981982[PubMed][Google Scholar]
  • 11. KassRSFreemanLCPotassium channels in the heart Cellular, molecular, and clinical implicationsTrends Cardiovasc Med19933149159[PubMed][Google Scholar]
  • 12. MacKinnonRDetermination of the subunit stoichiometry of a voltage-activated potassium channelNature1991350232235[PubMed][Google Scholar]
  • 13. SanguinettiMCJiangCCurranMEKeatingMTA mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channelCell199581299307[PubMed][Google Scholar]
  • 14. AbbottGWRameshBSraiSKSecondary structure of the MiRP1 (KCNE2) potassium channel ancillary subunitProtein Pept Lett2008156375[PubMed][Google Scholar]
  • 15. AbbottGWGoldsteinSASestiFDo all voltage-gated potassium channels use MiRPs?Circ Res200188981983[PubMed][Google Scholar]
  • 16. NerbonneJMKassRSMolecular physiology of cardiac repolarizationPhysiol Rev20058512051253[PubMed][Google Scholar]
  • 17. ZhangMJiangMTsengGN. minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel?Circ Res20018810121019[PubMed][Google Scholar]
  • 18. RoepkeTKTargeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K, slow1) and I(to, f)FASEB J20082236483660[PubMed][Google Scholar]
  • 19. DelponEFunctional effects of KCNE3 mutation and its role in the development of Brugada syndromeCirc Arrhythm Electrophysiol20081209218[PubMed][Google Scholar]
  • 20. EhrlichJRKvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currentsJ Biol Chem200427912331241[PubMed][Google Scholar]
  • 21. WuJKCNE2 modulation of Kv4.3 current and its potential role in fatal rhythm disordersHeart Rhythm20107199205[PubMed][Google Scholar]
  • 22. CoronelRElectrophysiological changes in heart failure and their implications for arrhythmogenesisBiochim Biophys Acta2013183224322441[PubMed][Google Scholar]
  • 23. JanseMJElectrophysiological changes in heart failure and their relationship to arrhythmogenesisCardiovasc Res200461208217[PubMed][Google Scholar]
  • 24. KaabSMolecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current densityCirculation19989813831393[PubMed][Google Scholar]
  • 25. GomezJFCardonaKRomeroLFerreroJMJr.TrenorBElectrophysiological and structural remodeling in heart failure modulate arrhythmogenesis. 1D simulation studyPLoS One20149e106602[PubMed][Google Scholar]
  • 26. WalmsleyJmRNA expression levels in failing human hearts predict cellular electrophysiological remodeling: a population-based simulation studyPLoS One20138e56359[PubMed][Google Scholar]
  • 27. FickerEDennisATWangLBrownAMRole of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERGCirc Res200392e87100[PubMed][Google Scholar]
  • 28. MasseyAJA novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cellsCancer Chemother Pharmacol201066535545[PubMed][Google Scholar]
  • 29. WenWLiuWShaoYChenLVER-155008, a small molecule inhibitor of HSP70 with potent anti-cancer activity on lung cancer cell linesExp Biol Med (Maywood)2014239638645[PubMed][Google Scholar]
  • 30. IwaiCHsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteinsCardiovasc Res2013100520528[PubMed][Google Scholar]
  • 31. ZhouZProperties of HERG channels stably expressed in HEK 293 cells studied at physiological temperatureBiophys J199874230241[PubMed][Google Scholar]
  • 32. PetreccaKAtanasiuRAkhavanAShrierAN-linked glycosylation sites determine HERG channel surface membrane expressionJ Physiol1999515Pt 14148[PubMed][Google Scholar]
  • 33. GongQAndersonCLJanuaryCTZhouZRole of glycosylation in cell surface expression and stability of HERG potassium channelsAm J Physiol Heart Circ Physiol2002283H7784[PubMed][Google Scholar]
  • 34. WalkerVEAtanasiuRLamHShrierACo-chaperone FKBP38 promotes HERG traffickingJ Biol Chem20072822350923516[PubMed][Google Scholar]
  • 35. CuiJAnalysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with KCNE2J Biol Chem20012761724417251[PubMed][Google Scholar]
  • 36. SchlichterLCRegulation of hERG and hEAG channels by Src and by SHP-1 tyrosine phosphatase via an ITIM region in the cyclic nucleotide binding domainPLoS One20149e90024[PubMed][Google Scholar]
  • 37. ZhouZGongQEpsteinMLJanuaryCTHERG channel dysfunction in human long QT syndrome. Intracellular transport and functional defectsJ Biol Chem19982732106121066[PubMed][Google Scholar]
  • 38. KupershmidtSDefective human Ether-a-go-go-related gene trafficking linked to an endoplasmic reticulum retention signal in the C terminusJ Biol Chem20022772744227448[PubMed][Google Scholar]
  • 39. AndersonCLLarge-scale mutational analysis of Kv11.1 reveals molecular insights into type 2 long QT syndromeNat Commun201455535[PubMed][Google Scholar]
  • 40. RotiECInteraction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-a-go-go-Related GeneJ Biol Chem20022774777947785[PubMed][Google Scholar]
  • 41. ZhangKPYangBFLiBXTranslational toxicology and rescue strategies of the hERG channel dysfunction: biochemical and molecular mechanistic aspectsActa Pharmacol Sin20143514731484[PubMed][Google Scholar]
  • 42. KaganAMelmanYFKrumermanAMcDonaldTV14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activityEMBO J20022118891898[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.