A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K+ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)

Dilshan Balasuriya; Lauren D'Sa; Ronel Talker; Elodie Dupuis; Fabrice Maurin; Patrick Martin; Franck Borgese; Olivier Soriani; J Michael Edwardson

doi:10.1074/jbc.M114.603506

. 2014 Sep 29;289(46):32353–32363. doi: 10.1074/jbc.M114.603506

A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K⁺ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)^*

Dilshan Balasuriya ^‡,¹, Lauren D'Sa ^‡, Ronel Talker ^‡, Elodie Dupuis ^§, Fabrice Maurin ^§, Patrick Martin ^¶, Franck Borgese ^¶, Olivier Soriani ^¶,², J Michael Edwardson ^‡,³

PMCID: PMC4231707 PMID: 25266722

Background: The sigma-1 receptor modulates the function of numerous ion channels.

Results: We studied the interaction between the sigma-1 receptor and hERG.

Conclusion: The interaction has a 4:1 stoichiometry and occurs at the plasma membrane.

Significance: The sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane.

Keywords: Atomic Force Microscopy (AFM), Fluorescence Resonance Energy Transfer (FRET), hERG, Molecular Imaging, Protein Complex, Sigma Receptor

Abstract

The sigma-1 receptor is an endoplasmic reticulum chaperone protein, widely expressed in central and peripheral tissues, which can translocate to the plasma membrane and modulate the function of various ion channels. The human ether-à-go-go-related gene encodes hERG, a cardiac voltage-gated K⁺ channel that is abnormally expressed in many human cancers and is known to interact functionally with the sigma-1 receptor. Our aim was to investigate the nature of the interaction between the sigma-1 receptor and hERG. We show that the two proteins can be co-isolated from a detergent extract of stably transfected HEK-293 cells, consistent with a direct interaction between them. Atomic force microscopy imaging of the isolated protein confirmed the direct binding of the sigma-1 receptor to hERG monomers, dimers, and tetramers. hERG dimers and tetramers became both singly and doubly decorated by sigma-1 receptors; however, hERG monomers were only singly decorated. The distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers had two peaks, at ∼90 and ∼180° in a ratio of ∼2:1, indicating that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Homogeneous time-resolved fluorescence (HTRF®) allowed the detection of the interaction between the sigma-1 receptor and hERG within the plane of the plasma membrane. This interaction was resistant to sigma ligands, but was decreased in response to cholesterol depletion of the membrane. We suggest that the sigma-1 receptor may bind to hERG in the endoplasmic reticulum, aiding its assembly and trafficking to the plasma membrane.

Introduction

The sigma receptor was initially classed as an opioid receptor (1), but is now known to be a distinct receptor with two subtypes: sigma-1 and sigma-2 (2). The sigma-1 receptor is a chaperone protein expressed in the CNS, liver, kidney, and heart (3, 4), and serves various functions as an inter-organelle signaling modulator. It also influences dendritic spine arborization (5) and controls Ca²⁺ release from the endoplasmic reticulum (ER)⁴ by regulating inositol 1,4,5-trisphosphate receptors (6).

The sigma-1 receptor is neuroprotective in stroke and cerebral ischemia (7), and has been linked to schizophrenia (8), Alzheimer disease (9), depression (10), and drug addiction (11). The receptor is up-regulated in tumors (12), and sigma-1 receptor agonists have been shown to inhibit neoplastic cell growth (13). The receptor also inhibits cell cycling in tumor cells, via K_v1.3 and Cl⁻ channels (14, 15), and it has been suggested that sigma-1 receptor antagonists may have chemotherapeutic properties (16).

The sigma-1 receptor has two transmembrane domains and no significant homology with any other mammalian protein (17). The N and C termini of the receptor were originally reported to be intracellular (18), although more recent evidence has indicated the opposite orientation (6, 19, 20). The C-terminal region is crucial to ligand and cholesterol binding (21, 22), and the receptor is activated by various exogenous ligands, including antipsychotic (e.g. haloperidol) and psychotomimetic (e.g. pentazocine) drugs (2). Recent evidence has implicated the hallucinogen N,N-dimethyltryptamine as a likely endogenous ligand (23). The sigma-1 receptor resides in the mitochondrion-associated ER membrane (6), but ligand binding or cellular stress can cause it to translocate to other sites (24), where it appears to have various roles.

The sigma-1 receptor is promiscuous in its modulation of ion channels, affecting voltage-gated (25 –30), ligand-gated (31 –33), volume-regulated (14), and acid-sensing (34) ion channels. A sigma-1 receptor·K_v1.4 interaction was shown to be independent of second messenger generation or phosphorylation, implying a direct interaction (35). In support of this idea, it was shown that the two proteins could be co-immunoprecipitated (18). Atomic force microscopy (AFM) imaging studies have demonstrated that the sigma-1 receptor binds to the acid-sensing ion channel-1a with 3-fold symmetry (36), to the Na_v1.5 channel with 4-fold symmetry (37), and to GluN1 but not GluN2A in the GluN1·GluN2A N-methyl-d-aspartate receptor (19). Interestingly, it has been shown recently that sigma-1 receptor activation results in an increase in trafficking of the NMDA receptor to the plasma membrane in the rat hippocampus (38).

The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the voltage-gated K⁺ ion channel, K_v11.1 (39). This channel is responsible for the rapid component of the delayed rectified K⁺ current, I_kr (40); it governs cardiac action potential duration at the plateau phase, and hence underlies cardiac repolarization (41, 42). Almost 200 hERG mutations have been identified, which cause misfolding and disrupted trafficking of the hERG protein, resulting in inherited long-QT syndrome (43 –45). Affected patients are also at risk of “torsades de pointes,” a fatal ventricular arrhythmia (40). hERG is also expressed in the brain (46), in smooth muscle (47), and in endocrine cells (48), and has been implicated in schizophrenia (46), similarly to the sigma-1 receptor (8). Furthermore, hERG is overexpressed in many tumors and cancer cell lines, notably leukemia, and controls cell migration and invasion via β1-integrin and VEGF-R1 (49), as well as conferring resistance to chemotherapy (50).

Co-immunoprecipitation of the sigma-1 receptor and hERG suggested a direct interaction between them (51). Further, the sigma-1 receptor was shown to potentiate hERG current density, indicating a functional interaction (51). Here, we set out to determine the nature of the interaction between the sigma-1 receptor and hERG. Using AFM imaging, we show that the sigma-1 receptor binds to assembled hERG channels with 4-fold symmetry, indicating that one sigma-1 receptor binds to each hERG subunit. Further, using homogeneous time-resolved fluorescence (HTRF®) technology, we demonstrate that the sigma-1 receptor and hERG interact at the plasma membrane and that this interaction is not altered by sigma ligands, but is reduced by cholesterol depletion.

EXPERIMENTAL PROCEDURES

Cell Culture

tsA 201 cells (a subclone of HEK-293 cells stably expressing the SV40 large T-antigen) and HEK-293 cells stably transfected with hERG bearing a HA tag in the extracellular loop between residues 443–444 (hE(HA)RG), and the human sigma-1 receptor bearing a Myc tag at either the N terminus (Myc-Sigma) or the C terminus (Sigma-Myc), were grown in DMEM supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, in an atmosphere of 5% CO₂/air.

Constructs

The following constructs were used. To create Sigma-FLAG, cDNA encoding the human sigma-1 receptor, with a C-terminal FLAG epitope tag, was subcloned into the vector pcDNA3.1/V5-His using HindIII and AgeI so as to delete the V5 epitope tag but leave the His₆ tag. (The His₆ tag was not used in any of the experiments described here.) To create Myc-SigmaHalo, a HaloTag® was fused to the C terminus of the sigma-1 receptor bearing an N-terminal Myc tag. This construct was inserted into a puromycin-resistant retroviral bicistronic expression vector (52). To create Myc-SigHaloMa, steps were followed as above, but with the HaloTag® inserted between residues 60–61 of the sigma-1 receptor construct. To create hE(HA)RG, the DraIII-BamH1 fragment of a pcDNA-Zeo construct containing hERG bearing an HA tag between residues 443–444 (i.e. as in the stably transfected HEK-293 cells described above) was subcloned into the pPRIHy retroviral vector (52). To create hERG-HA, hERG bearing a C-terminal HA tag was subcloned into a hygromycin-resistant retroviral bicistronic expression vector (52). Sequences of all constructs were verified before use.

Transient Transfection of tsA 201 Cells

Transient transfections of tsA 201 cells with DNA encoding Sigma-FLAG were carried out using the calcium phosphate precipitation method. A total of 250 μg of DNA was used to transfect cells in 5 × 162-cm² culture flasks. After transfection, cells were incubated for 48 h at 37 °C to allow protein expression.

Immunofluorescence

Protein expression and intracellular localization were checked using immunofluorescence analysis of small-scale cultures. Cells were fixed, permeabilized, and incubated with rabbit polyclonal anti-HA (Sigma, H6908), mouse monoclonal anti-Myc (Life Technologies, R950-25), or mouse monoclonal anti-FLAG (Sigma) primary antibodies followed by appropriate FITC- or Cy3-conjugated secondary antibodies (Sigma). Cells were imaged by confocal laser scanning microscopy.

In Situ Proximity Ligation Assay

HEK-293 cells stably expressing hE(HA)RG and either Myc-Sigma or Sigma-Myc, growing on lysine- and collagen-coated glass coverslips, were subjected to the proximity ligation reaction (53), according to the manufacturer's instructions (Olink Bioscience). Antibodies used were rabbit polyclonal anti-HA plus either mouse monoclonal anti-Myc, or as a negative control, mouse monoclonal anti-V5 (Life Technologies, R960-25). Cells were imaged by confocal laser scanning microscopy.

Solubilization and Purification of Epitope-tagged Proteins

Cells were solubilized in 1% (v/v) Triton X-100 for 1 h, before centrifugation at 61,000 × g to remove insoluble material. On one occasion, a sample of this detergent extract was heated to 100 °C for 10 min and then incubated with N-glycosidase F (New England Biolabs) at 37 °C for 2 h. For immunoisolation of proteins, the solubilized extract was incubated with anti-Myc- or anti-FLAG-agarose beads (Sigma), as appropriate, for 3 h. The beads were washed extensively, and bound proteins were eluted with either Myc or triple-FLAG peptide (100 μg/ml; Sigma). Samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining and/or immunoblotting, using mouse monoclonal antibodies against HA or FLAG. Immunoreactive bands were visualized using a horseradish peroxidase-conjugated goat anti-mouse secondary antibody followed by enhanced chemiluminescence.

AFM Imaging of Isolated Proteins

Isolated protein samples were diluted to a final concentration of 0.04 nm, and 45 μl of the sample was allowed to adsorb to freshly cleaved mica disks. After a 5-min incubation, the sample was washed with Biotechnology Performance Certified-grade water (Sigma) and dried under nitrogen. Imaging was performed with a Bruker Multimode atomic force microscope controlled by a NanoScope IIIa controller. Samples were imaged in air, using tapping mode. The silicon cantilevers used had a drive frequency of ∼300 kHz and a specified spring constant of 40 newtons/m (Olympus). The applied imaging force was kept as low as possible (A_s/A₀ ∼0.85).

For individual sigma-1 receptor particles, molecular volumes were determined using Scanning Probe Image Processor Version 5 (Image Metrology). It is well known that the geometry of the scanning AFM probe introduces a tendency to overestimate particle diameter. To minimize this probe convolution error, we used a particle threshold of 0.1 nm to provide accurate measurements of diameter. For particles within complexes, particle heights and diameters were measured manually using the NanoScope software and used to calculate molecular volumes, according to the equation

where h is the particle height and r is the radius (54). This equation assumes that the adsorbed particles adopt the form of a spherical cap.

Molecular volume based on molecular mass was calculated using the equation

where M₀ is the molecular mass, N₀ is Avogadro's number, V₁ and V₂ are the partial specific volumes of particle (0.74 cm³/g) and water (1 cm³/g), respectively, and d is the extent of protein hydration (taken as 0.4 g of water/g of protein).

Selection of Binding Events

Several criteria were used to identify sigma-1 receptor·hERG complexes. Heights and radii were measured for all particles, and the particle volumes were calculated. Bound particles needed to have a molecular volume between 30 and 120 nm³; that is, between half and double the peak volume of isolated sigma-1 receptors (see below). A cross-section was drawn through the junction between the sigma-1 receptor and the adjacent hERG particle, and the height of the lowest point between the two proteins was measured. This height needed to be greater than 0.3 nm for the sigma-1 receptor to be considered bound. Any particle was rejected if its length was greater than twice its width. To be considered a double binding event, all particles and both binding events needed to meet all the above criteria.

Statistical Analysis

Histograms were drawn with bin widths chosen according to Scott's equation.

where σ is an estimate of the standard deviation and n is the sample size (55). Where Gaussian curves were fitted to the data, the number of curves was chosen so as to maximize the r² value while giving significantly different means using Welch's t test for unequal sample sizes and unequal variances (56).

Extracellular HTRF®

To monitor the proximity between the sigma-1 receptor and hERG, we used various combinations of two hERG constructs with either an extracellular (hE(HA)RG) or an intracellular (hERG-HA) HA tag and two sigma-1 receptor constructs with either an extracellular (Myc-SigmaHalo) or an intracellular (Myc-SigHaloMa) HaloTag®.

HTRF® measurements were performed on transfected HEK-293 cells using Tag-lite® reagents. Tb²⁺ cryptate-linked HaloTag® specific substrate (HaloTag-Lumi4®-Tb SHALOTBC) and d2-conjugated anti-HA (mAb anti-HA-d2, 610HADAB) or anti-Myc (mAb anti-Myc-d2, 61MYCDAB) antibodies were used as energy donor and acceptor, respectively. Cells, in 96-well plates, were transiently transfected with the various plasmids (50 ng of hERG DNA plus 150 ng of the sigma-1 receptor DNA per well) using Lipofectamine 2000. Thirty-six hours after transfection, cells were incubated in Tag-lite® labeling medium (LABMED) containing HaloTag-Lumi4®-Tb and mAb anti-HA-d2 at the indicated concentrations. Cells were incubated for 90 min at 37 °C to allow the HaloTag-Lumi4®-Tb substrate to bind covalently to the HaloTag®, and then the fluorescence of Tb²⁺ cryptate and d2, at 620 nm and 665 nm, respectively, was measured (without washing) 100 μs after excitation at 340 nm using a Synergy 4 (BioTek, Winooski, VT) HTRF®-compatible instrument. HTRF® signals were expressed as %(ΔF) = ((665/620)_sample − (665/620)_blank) × 100/(665/620)_blank) or HTRF® ratio ((665/620)_sample × 10,000). For detection of proteins at the cell surface, we measured cryptate emission at 620 nm following excitation at 340 nm of mAb anti-HA-Tb (610HATAB), mAb anti-Myc-Tb (61MYCTAB), or HaloTag-Lumi4®-Tb in a time-resolved mode. Tag-lite® technology and reagents were supplied by CisBio Bioassays, Codolet, France.

RESULTS

In an initial experiment, tsA 201 cells were transfected with DNA encoding Sigma-FLAG. Immunofluorescence analysis, using an anti-FLAG primary antibody followed by a Cy3-conjugated secondary antibody, revealed the presence of the protein throughout the cell cytoplasm (Fig. 1A). Transfected cells were solubilized in 1% Triton X-100 detergent, and sigma-1 receptor-FLAG was isolated by anti-FLAG immunoaffinity chromatography. A Coomassie Blue-stained gel of the isolated protein (Fig. 1B) shows a single major band at a molecular mass of 33 kDa. The isolated protein was also analyzed by immunoblotting using an anti-FLAG antibody (Fig. 1C). A single immunopositive band, again at 33 kDa, was seen in both the total detergent extract and the protein eluted from the immunobeads. Hence, Sigma-FLAG was successfully isolated from the transfected cells.

FIGURE 1. — **Isolation and analysis of sigma-1 receptors from transiently transfected tsA 201 cells.** A, immunofluorescence detection of Sigma-FLAG. Cells were fixed, permeabilized, and incubated with a mouse monoclonal anti-FLAG primary antibody followed by a Cy3-conjugated goat anti-mouse secondary antibody. Cells were imaged by confocal laser scanning microscopy. *Scale bar*, 50 μm. B, Coomassie Blue stain of immunoisolated Sigma-FLAG (*left lane*, Sigma-FLAG indicated by *arrow*). Molecular mass markers (kDa) are shown in the *right lane. C*, anti-FLAG immunoblot showing total (T) and eluted (E) fractions from the immunoisolation. D, low-magnification AFM image of isolated sigma-1 receptors. *Scale bar*, 200 nm; *color-height scale*, 0–3 nm. E, frequency distribution of molecular volumes of sigma-1 receptor particles. The curve indicates the fitted Gaussian function. r² = 0.93; p < 0.001. The peak of the distribution (±S.E.) is indicated.

Low-magnification AFM images of isolated sigma-1 receptors revealed a relatively homogeneous distribution of particles (Fig. 1D). The molecular volumes of a number of these particles were calculated; a frequency distribution of the volumes (Fig. 1E) had a single peak, at 56 ± 2 (S.E.) nm³ (n = 480), close to the expected volume of 63 nm³ for a sigma-1 receptor, of molecular mass 33 kDa, according to Equation 2. Hence, the imaged particles represent individual sigma-1 receptors.

Immunofluorescence analysis of HEK-293 cells stably expressing hE(HA)RG and Myc-Sigma, using anti-HA and anti-Myc primary antibodies, revealed that all cells expressed both proteins and that the proteins were distributed throughout the cell cytoplasm (Fig. 2A). Immunoblotting of a total cell extract showed that hE(HA)RG migrated as a doublet of bands at molecular masses of ∼130 and ∼150 kDa (Fig. 2B), whereas the sigma-1 receptor ran as a single major band at ∼33 kDa, with a fainter band at ∼60 kDa, which likely represents a dimer (Fig. 2C). We speculated that the pair of bands given by hE(HA)RG might result from the presence of unglycosylated and glycosylated forms, as shown previously (45), which would represent ER and post-ER species. To test this idea, we treated the cell extract with N-glycosidase F, which should remove all N-linked oligosaccharides. Now the protein migrated as a single band at ∼130 kDa, confirming that this form is indeed unglycosylated (Fig. 2B).

FIGURE 2. — **Analysis of sigma-1 receptor/hERG expression and interaction in stably transfected HEK-293 cells.** A, immunofluorescence detection of hE(HA)RG and Myc-Sigma. Cells were fixed, permeabilized, and incubated with rabbit polyclonal anti-HA and mouse monoclonal anti-Myc primary antibodies followed by Cy3-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse secondary antibodies. Cells were imaged by confocal laser scanning microscopy. *Scale bar*, 50 μm. B, anti-HA immunoblot of a total cell extract showing hE(HA)RG before and after treatment with N-glycosidase F. Molecular mass markers (kDa) are shown at the *right. C*, anti-Myc immunoblot of a total cell extract showing Myc-Sigma. D, *in situ* proximity ligation assay between the extracellular HA tag on hERG and the Myc tag on either the N terminus (N) or the C terminus (C) of the sigma-1 receptor. The constructs used in the assay are illustrated in the *upper panel* (*green star*, HA tag; *red circle*, Myc tag). Cells were fixed, permeabilized, and incubated with rabbit polyclonal anti-HA and mouse monoclonal anti-Myc antibodies. The proximity ligation reaction was then carried out (*lower panels*). In the control experiment, using cells expressing hE(HA)RG plus Myc-Sigma, an anti-V5 antibody, replaced the anti-Myc antibody. *Scale bar*, 50 μm.

To determine whether the sigma-1 receptor and hERG interact within the stably transfected cells, we used an in situ proximity ligation assay. The assay (53) uses two secondary antibodies, each bearing a short DNA strand. When the secondary antibodies are brought into close proximity (<40 nm) by binding to their relevant primary antibodies (in this case rabbit polyclonal anti-HA and mouse monoclonal anti-Myc), the DNA strands hybridize with an additional circle-forming oligodeoxynucleotide. Ligation then creates a complete circularized oligodeoxynucleotide, and rolling circle amplification increases the amount of circular DNA several hundredfold. The DNA is then visualized using a fluorescent probe. The constructs used in the assay are illustrated in Fig. 2D (upper panel). The in situ proximity ligation assay gave a bright signal with cells expressing hERG together with either Myc-Sigma or Sigma-Myc, but not when a mouse monoclonal anti-V5 control antibody was used instead of anti-Myc (Fig. 2D, lower panel). This result indicates that the two proteins do indeed come into close proximity within the cells, irrespective of the position of the Myc tag on the sigma-1 receptor. Further, given that the HA tag on hERG is extracellular, we can conclude that both the N terminus and the C terminus of the sigma-1 receptor are also extracellular, in agreement with several previous studies (6, 19, 20).

Protein was isolated from cells co-expressing Myc-Sigma and hE(HA)RG by anti-Myc immunoaffinity chromatography. The isolated sample was analyzed by immunoblotting with anti-Myc and anti-HA antibodies. The anti-Myc antibody detected a single band at 33 kDa in the eluted sample (Fig. 3A, left panel), demonstrating the presence of the sigma-1 receptor. The anti-HA blot showed bands at ∼130 and ∼150 kDa in the total detergent extract, with the ∼150-kDa band being the stronger of the two (Fig. 3A, right panel). In contrast, in the eluted sample, the ∼130-kDa band was by far the stronger. This result demonstrates that hE(HA)RG can be co-isolated from the cells with Myc-Sigma and that although some glycosylated (mature) hERG binds to the sigma-1 receptor, the unglycosylated (ER) form of hERG is isolated preferentially.

FIGURE 3. — **Direct binding of the sigma-1 receptor to hERG.** A, protein was isolated from cells expressing Myc-Sigma and hE(HA)RG by anti-Myc immunoaffinity chromatography. Total (T) and eluted (E) proteins were detected by immunoblotting using anti-Myc or anti-HA antibodies. Molecular mass markers (kDa) are shown at the *right. B*, low-magnification AFM images of proteins isolated from cells expressing Myc-Sigma and hE(HA)RG. *Arrows* indicate hERG tetramers singly (*left panel*), doubly (*center panel*), or triply (*right panel*) decorated by sigma-1 receptors. *Scale bar*, 200 nm; *color-height scale*, 0–3 nm. C, frequency distribution of molecular volumes of peripheral particles attached to a larger central particle. The curve indicates the fitted Gaussian function. r² = 0.94; p < 0.0001. The mean of the distribution (±S.E.) is indicated. D, frequency distribution of volumes of the central particles decorated by one or more sigma-1 receptors. The curve indicates the fitted Gaussian functions. r² = 0.99; p < 0.0001 for all three peaks. The means of the distribution (±S.E.) are indicated. E, frequency distribution of volumes of doubly decorated central particles. The curve indicates the fitted Gaussian function. r² = 0.91; p < 0.0001 for both peaks. The means of the distribution (±S.E.) are indicated.

Low-magnification AFM images of co-isolated sigma-1 receptor and hE(HA)RG showed a population of large particles, some of which were decorated by one, two, or three smaller particles (Fig. 3B). A frequency distribution of volumes of the smaller particles, calculated according to Equation 1 (Fig. 3C), had a single peak at 60 ± 2 nm³ (n = 641), very similar to the peak volume for sigma-1 receptors alone (Fig. 1E) and to the expected volume of 63 nm³. Hence, the small bound particles are very likely to be sigma-1 receptors.

We set a volume range of 30–120 nm³ for the smaller bound particles (i.e. between half and double the peak volume) and then measured the volumes of the larger particles in all decoration states (i.e. single, double, and triple). A frequency distribution of these volumes had three peaks, at 313 ± 6, 520 ± 11, and 900 ± 27 nm³ (n = 485; Fig. 3D). The expected molecular volume for a hERG monomer of molecular mass ∼130 kDa is 246 nm³; hERG dimers and tetramers would therefore have volumes of 492 and 984 nm³, respectively. The three volume peaks, therefore, likely represent hERG monomers, dimers, and tetramers. Interestingly, a frequency distribution of volumes of doubly decorated larger particles had only two peaks, at 482 ± 11 and 950 ± 55 nm³ (n = 78), corresponding to hERG dimers and tetramers, respectively (Fig. 3E). The smallest peak seen in Fig. 3D, corresponding to monomers, is absent, indicating that each subunit is able to bind only one sigma-1 receptor molecule.

Based on the hERG volume distribution, volume ranges were set for the various assembly states; specifically, particles in the volume ranges 200–400, 400–650, and 700–1300 nm³ were assumed to be monomers, dimers, and tetramers, respectively. The 650–700 nm³ volume range fell inconclusively between dimers and tetramers and so was not included in subsequent analyses. Using these volume ranges, it was possible to show that 34% of all sigma-1 receptor/hERG complexes involved hERG monomers; 33% involved dimers; and 26% involved tetramers.

Zoomed images of singly decorated hERG monomers are shown in Fig. 4A, and singly and doubly decorated hERG dimers and tetramers are shown in Fig. 4B and C, respectively. As can be seen, double decoration of tetramers occurred at angles of ∼90 and ∼180°. We identified tetramers that had been decorated by two sigma-1 receptors and measured the angles between the bound receptors. This was done in each case by joining the highest point on the central particle (the hERG tetramer) to the highest points on the peripheral particles (the sigma-1 receptors) by lines and then determining the angle between the two lines. A frequency distribution of the angles is shown in Fig. 4D. The distribution has two peaks: a large peak at 85 ± 3° and a smaller peak at 170 ± 35° (n = 58); the ratio of the numbers of particles in the two peaks (defined as <120 and >120°) is 2.2:1. This angle profile, with two peaks at around 90 and 180°, in a ratio of ∼2:1 suggests that the hERG tetramer presents four perpendicular binding sites to the sigma-1 receptor and that these are randomly occupied.

FIGURE 4. — **Sigma-1 receptor decoration of hERG monomers, dimers, and tetramers.** A, zoomed images showing particles from the lowest volume (200–400 nm³) hERG peak in Fig. 3D (monomers) singly decorated by sigma-1 receptors. *Scale bar*, 20 nm; *color-height scale*, 0–2 nm. B, zoomed images showing particles from the middle-volume (400–650 nm³) hERG peak (dimers) singly (*upper panels*) and doubly (*lower panels*) decorated by sigma-1 receptors. *Scale bar*, 20 nm; *color-height scale*, 0–2 nm. C, zoomed images showing particles from the largest volume (700–1300 nm³) hERG peak (tetramers) singly decorated by sigma-1 receptors (*top panels*) and doubly decorated at either ∼90° (*center panels*) or ∼180° (*bottom panels*). *Scale bar*, 20 nm; *color-height scale*, 0–2 nm. D, frequency distribution of angles between pairs of sigma-1 receptors bound to hERG tetramers. The curve indicates the fitted Gaussian functions. r² = 0.97; p < 0.0001 for the 85° peak and 0.016 for the 170° peak. The means of the distribution (±S.E.) are indicated.

To be sure that the protein complexes reported above represented genuine sigma-1 receptor·hERG interactions, we compared the numbers of binding events observed when the sigma-1 receptor was isolated from cells co-expressing hERG with those seen when the sigma-1 receptor was expressed alone. The two samples were prepared so as to have approximately equal particle densities on the mica supports. We then analyzed 212 2 × 2 μm-AFM images from each sample. For the sigma-1 receptor·hERG sample, these images contained 449 binding events involving all hERG assembly states, as compared with 54 events for the sigma-1 receptor-only sample. Further, there were 49 multiple binding events in the sigma-1 receptor·hERG sample, but none in the sigma-1 receptor-only sample. We are confident, therefore, that the vast majority of the observed binding events, and in particular the multiple binding events, do indeed represent genuine sigma-1 receptor·hERG interactions. The background binding events seen in the sigma-1 receptor-only sample might represent interactions of the sigma-1 receptor with unidentified endogenous binding partners.

To test whether the sigma-1 receptor and hERG interact within the plane of the plasma membrane, we developed an HTRF® approach (57), combining standard Förster resonance energy transfer (FRET) technology with time-resolved measurement of fluorescence, thus eliminating short-lived background fluorescence (57). The constructs used for HTRF® are illustrated schematically in Fig. 5A. The sigma-1 receptor, bearing a Myc tag at its N terminus, had either an extracellular (Myc-SigmaHalo) or an intracellular (Myc-SigHaloMa) HaloTag®, a modified haloalkane dehalogenase designed to bind covalently to synthetic substrates (58). Similarly, hERG had either an extracellular (hE(HA)RG) or an intracellular (hERG-HA) HA tag. The assay relies on an interaction between the HaloTag-Lumi4®-Tb and a mAb anti-HA-d2, as illustrated in Fig. 5B. Emissions at 620 nm (donor) are used as an internal reference, while emissions at 665 nm (acceptor) serve as an indicator of the reaction being assessed. The measurement of emissions at two different wavelengths allows the ratiometric presentation of data. Significantly, time-resolved FRET (TR-FRET) is not observed when the two fluorophores are located on opposite sides of the membrane. As shown in Fig. 5C, we could detect a clear TR-FRET signal that increased with the concentration of mAb anti-HA-d2, when HEK-293 cells co-expressed Myc-SigmaHalo and hE(HA)RG, but only a weak (background) signal in untransfected cells. Cells were transfected with various combinations of the four constructs, and the expression of each construct was assessed by immunoblotting (Fig. 5D). After incubation of cells with terbium-conjugated anti-HA tag, a 620-nm emission signal was observed with hE(HA)RG but not with hERG-HA, as expected from the location of the tags (Fig. 5E). Similarly, the HaloTag-Lumi4®-Tb gave a signal for Myc-SigmaHalo but not for Myc-SigHaloMa (Fig. 5F). Expression of both sigma-1 receptor constructs at the plasma membrane was confirmed using a terbium-conjugated anti-Myc antibody, which gave very similar 620-nm emission signals in both cases (Fig. 5G).

FIGURE 5. — **Characterization of the constructs used for the HTRF® assays.** A, diagram illustrating the positions of the tags on the various constructs (*green star*, HA tag; *red circle*, Myc tag; *yellow hexagon*, HaloTag®). N, N terminus; C, C terminus. B, diagram illustrating the principle underlying the assay. Extracellular TR-FRET occurs between Myc-SigmaHalo labeled with HaloTag-Lumi4®-Tb (donor) and hE(HA)RG labeled with mAb anti-HA-d2 (acceptor). C, TR-FRET signals between the indicated pair of proteins transiently expressed in HEK-293 cells. Cells were incubated in Tag-lite® labeling medium containing 1 nm HaloTag-Lumi4®-Tb and varying concentrations of mAb anti-HA-d2. Each point represents the median of five experiments performed in triplicate ± the first and third quartiles. The means of the distribution (±S.E.) are indicated. D, immunoblots of extracts of transiently transfected HEK-293 cells expressing the four constructs shown in *A. E–G*, detection of the constructs via terbium-cryptate emission at 620 nm for MAb anti-HA-Tb (E), HaloTag-Lumi4®-Tb (F), and MAb anti-Myc-Tb (G). The means of the distribution (±S.E.) are indicated.

We first tested whether sigma-1 receptor expression affected the levels of hERG at the plasma membrane. As shown in Fig. 6A, we found that when Myc-SigmaHalo was co-expressed with hE(HA)RG, emission at 620 nm was significantly increased as compared with the emission measured with hE(HA)RG alone, demonstrating that the sigma-1 receptor does indeed potentiate hERG plasma membrane expression. This result is in a good agreement with previous studies suggesting that sigma-1 receptor potentiates the intracellular trafficking of various ion channels (37, 38, 51). We then looked for an interaction between the sigma-1 receptor and hERG at the plasma membrane. Cells were transfected with three pairs of constructs. As shown in Fig. 6B, TR-FRET was observed only when the tags on the two proteins were extracellular (i.e. with Myc-SigmaHalo plus hE(HA)RG).

FIGURE 6. — **Demonstration and characterization of a direct interaction between the sigma-1 receptor and hERG in the plasma membrane.** A, detection of hE(HA)RG via terbium-cryptate emission at 620 nm for terbium-conjugated mAb anti-HA-Tb in cells expressing either hE(HA)RG alone or hE(HA)RG plus Myc-SigmaHalo. B, TR-FRET signals between the indicated constructs transiently expressed in HEK-293 cells. Cells were incubated in Tag-lite® labeling medium containing 1 nm HaloTag-Lumi4®-Tb and 8 nm mAb anti-HA-d2. C, effects of sigma-1 receptor ligands on TR-FRET. (+*)PTZ* = (+)phenothiazine. D, effect of methyl-β-cyclodextrin (MβCD) on TR-FRET. Data are Tukey's boxplots of three (A) or five (*B–D*) experiments performed in triplicate ± the first and third quartiles (*bar*, median; *filled diamond*, first quartile; *spiked symbol*, third quartile). *, p < 0.05, bidirectional Kruskal and Wallis test; NS, nonsignificant. The means of the distribution (±S.E.) are indicated.

We next examined whether the sigma-1 receptor ligands igmesine, (+)pentazocine, and BD 1047 (59) affected the interaction of the sigma-1 receptor with hERG. The concentration ranges chosen for each compound were based on those reported previously to give rise to sigma-1 receptor-dependent modulation of various ion channels (6, 15, 27, 37, 38), including hERG (51). As shown in Fig. 6C, none of these ligands had any effect on the TR-FRET signal.

It is known that the sigma-1 receptor possesses a sterol binding pocket and is colocalized with cholesterol- and neutral lipid-rich microdomains at the mitochondrion-associated ER membrane (6). We therefore tested the sensitivity of the TR-FRET reaction to the membrane cholesterol content. Cells were incubated with the cholesterol chelator methyl-β-cyclodextrin over a concentration range shown previously to remove cholesterol from cell membranes and to perturb receptor-mediated endocytosis (60). As shown in Fig. 6D, methyl-β-cyclodextrin at 10 mm reduced the TR-FRET signal by 27%, but lower concentrations had no significant effect.

DISCUSSION

We have shown here that the sigma-1 receptor and hERG interact within co-transfected cells and that the two proteins can be co-isolated by immunoaffinity chromatography. Further, AFM imaging of the isolated proteins confirmed that the sigma-1 receptor·hERG interaction is direct. Interestingly, unglycosylated hERG, rather than the more abundant glycosylated form, was the predominant species co-isolated with the sigma-1 receptor. Given that initial glycosylation of hERG occurs in the ER, with further glycosylation in the Golgi (61), the direct binding of the sigma-1 receptor to immature hERG likely occurs predominantly in the ER, consistent with the existence of a major intracellular sigma-1 receptor pool in this compartment (6). We also show that hERG expression at the plasma membrane is enhanced by co-expression of the sigma-1 receptor. Caution should of course be exercised in drawing general conclusions based on experiments involving exogenous overexpression of proteins. However, the fact that similar effects of the sigma-1 receptor on the behavior of hERG have been reported previously for K562 myeloid leukemia cells (51), which endogenously express both proteins, gives us confidence in the physiological relevance of our findings.

Heterologous sigma-1 receptor expression has been shown previously to increase hERG current density, without influencing the activation/inactivation parameters of the channel (51). The mechanism underlying this effect does not involve a sigma-1 receptor-mediated effect on hERG transcription because sigma-1 receptor silencing had no effect on hERG mRNA production in K562 cells (51). Interestingly, sigma-1 receptor silencing significantly reduced the amount of mature hERG in cells, whereas the amount of immature hERG increased (51). This effect, along with our demonstration of a direct interaction, suggests that the sigma-1 receptor increases hERG current density by potentiating hERG maturation, perhaps by assisting the assembly and folding of immature hERG subunits. This hypothesis is supported by the fact that co-expression of the sigma-1 receptor with hERG resulted in higher channel detection at the plasma membrane. In good agreement with this idea, reduced hERG maturation was seen when cells were treated with inhibitors of Hsp90, a known hERG chaperone (62). Functional studies have shown that the sigma-1 receptor increases the stability of mature hERG and decreases membrane recycling of the ion channel (51). Our demonstration of an interaction of the sigma-1 receptor with mature hERG is consistent with this effect.

The hERG channel is built from four identical hERG subunits and therefore has 4-fold symmetry. We have shown that each hERG subunit presents a binding site for one sigma-1 receptor and that these sites are occupied randomly. A similar result was reported recently for sigma-1 receptor binding to the Na_v1.5 channel (37), although in this latter case a channel with pseudo-4-fold symmetry is generated by a single polypeptide. In the case of hERG, sigma-1 receptor-decorated monomers and dimers were seen, along with fully assembled tetramers. Indeed, tetramers accounted for only about one-quarter of the decorated species. This relative scarcity of decorated tetramers might indicate the presence of a significant intracellular pool of incompletely assembled channels, or perhaps more likely, extensive disassembly of the channels during isolation, when the channel is exposed to detergent for several hours. Interestingly, there is no molecular volume peak corresponding to decorated trimers, suggesting that if the channel is indeed falling apart during isolation, it does so by dissociating into dimers, which in turn dissociate into monomers.

AFM imaging has demonstrated interactions with acid-sensing ion channel-1a (36), Na_v1.5 (37), and the GluN1 subunit of the GluN1·GluN2A NMDA receptor (19), although the intracellular locations of these interactions are unclear. Previously reported effects of the sigma-1 receptor on ion channel properties (25 –35) suggested an interaction at the plasma membrane, but the possibility that the sigma-1 receptor resided in a membrane compartment just beneath the plasma membrane could not be ruled out. In the present study, we used HTRF® to determine whether the sigma-1 receptor interacts with hERG in the plane of the plasma membrane. Because all of the compounds involved in the HTRF® reaction are membrane-impermeant, TR-FRET can be used to evaluate the hERG·sigma-1 interaction on the external side of the plasma membrane. Our results show that the interaction revealed in protein extracts by AFM is also detectable at the plasma membrane of intact cells. To our knowledge, this is the first time that a direct interaction between the sigma-1 receptor and an ion channel has been demonstrated at the cell surface. We have previously reported that the sigma-1 receptor acts as a hERG chaperone protein, increasing the efficiency of its maturation (51). We now show that this interaction is maintained at the plasma membrane, in line with the observation that the sigma-1 receptor increases the stability of the fully glycosylated (mature) form of the channel (51), which represents the population located at cell surface.

We have shown previously that the sigma-1 receptor binds to the tetrameric Na_v1.5 with 4-fold symmetry and to the trimeric ASIC1 with 3-fold symmetry (36, 37). Here, we show that the sigma-1 receptor interacts with hERG with 4-fold symmetry. Taking into account the fact that the sigma-1 receptor can be co-immunoprecipitated with a truncation mutant of K_v1.3 devoid of the N- and C-terminal regions of the protein, we speculate that the sigma-1 receptor interacts with the transmembrane regions of its target proteins (30). A recent study described the chaperone domain of the sigma-1 receptor (61). This strongly amphipathic domain (residues 176–204) was proposed to anchor the chaperone domain to membranes. It contains two cholesterol recognition motifs, which are also involved in haloperidol and cocaine binding, and one helix implicated in membrane association (63). Our current observation that the FRET signal between the sigma-1 receptor and hERG is sensitive to a decrease in the cholesterol content of the plasma membrane is consistent with previous data indicating that lipids influence the behavior of the sigma-1 receptor (22).

It should be borne in mind that the link between the ligand binding sites of the sigma-1 receptor and the cholesterol recognition motifs is still unclear. The association between the sigma-1 receptor and ASIC1a was reduced by ∼50% by haloperidol, and the association between the sigma-1 receptor and Na_v1.5 was reduced by ∼80% by pentazocine (36, 37), prompting speculation that the action of sigma ligands might involve displacement of cholesterol from the sigma-1 receptor. In contrast, in the present study, igmesine, (+)pentazocine, and BD 1047 were all unable to modify the interaction between the sigma-1 receptor and hERG. This result is consistent with our previous observation that co-immunoprecipitation of the sigma-1 receptor and hERG was not disrupted by igmesine (51). It should be emphasized that we have not directly demonstrated that the ligands do indeed bind to the sigma-1 receptor. However, although it is possible, for example, that the addition of the epitope tags might have interfered with the ligand binding site, we suggest that this is unlikely given that the tags were added to the N and C termini of the protein, far away from the ligand binding site.

A wide range of effects of sigma-1 receptor ligands has been reported, depending on the nature of the sigma-1 receptor partner and the tissue in which the sigma-1 receptor is expressed. For example, igmesine and (+)pentazocine cause the dissociation of the sigma-1 receptor from the inositol 1,4,5-trisphosphate receptor in the endoplasmic reticulum (4), whereas cocaine strengthens the interaction between the sigma-1 receptor and the K_v1.2 channel in the mouse nucleus accumbens (20). Further, cocaine promotes the formation of sigma-1 receptor/dopamine D1 receptor complexes (64) but interacts with sigma-1 receptor/D2 heteromers to inhibit downstream signaling (65). Further studies are clearly needed to characterize the interactions between the sigma-1 receptor and its partners and to elucidate the mechanisms of action of sigma-1 receptor ligands and cholesterol.

Our study adds the hERG channel to a growing cohort of channels to which the sigma-1 receptor is known to bind directly. Given the involvement of hERG in cancer cell migration and invasiveness, as well as in cardiac arrhythmias, a clearer understanding of the molecular basis of the effects of the sigma-1 receptor may pave the way for a therapeutic approach based on modulation of this interaction.

This work was supported by Kidney Research UK (to J. M. E.), the CNRS, the University of Nice Sophia Antipolis, and the Association Ti'Toine Normandie (to O. S.). Elodie Dupuis and Fabrice Maurin are employees of CisBio Assays, the company that supplied the reagents used in the homogenous time-resolved fluorescence assays.

⁴

The abbreviations used are:

ER: endoplasmic reticulum
AFM: atomic force microscopy
hERG: human ether-à-go-go-related gene
HTRF®: homogeneous time-resolved fluorescence
FRET: Förster resonance energy transfer
TR-FRET: time-resolved FRET.

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PERMALINK

A Direct Interaction between the Sigma-1 Receptor and the hERG Voltage-gated K⁺ Channel Revealed by Atomic Force Microscopy and Homogeneous Time-resolved Fluorescence (HTRF®)^*

Dilshan Balasuriya

Lauren D'Sa

Ronel Talker

Elodie Dupuis

Fabrice Maurin

Patrick Martin

Franck Borgese

Olivier Soriani

J Michael Edwardson

Abstract

Introduction