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®)

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.

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

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.

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.

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.

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.

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.

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.