Demonstration of a direct interaction between sigma-1 receptors and acid-sensing ion channels

Abstract

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His(6)-tagged sigma-1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131 degrees . When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134 degrees , confirming that the small particles were sigma-1 receptors. Of interest, we found that the sigma-1 receptor ligand haloperidol caused an approximately 50% reduction in ASIC1a/sigma-receptor binding, suggesting a way in which sigma-1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the sigma-1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 sigma-1 receptor/1 ASIC1a subunit.

Figure 1

Isolation of proteins from ASIC1a-expressing cells. (A) Immunofluorescence detection of ASIC1a in stably transfected HEK-293 cells. Cells were fixed, permeabilized, and incubated with either rabbit polyclonal anti-ASIC1a antibody or mouse monoclonal anti-His₆ antibody, followed by Cy3-conjugated goat anti-rabbit or anti-mouse secondary antibodies, as appropriate. Cells were imaged by confocal laser scanning microscopy. (B) Immunofluorescence detection of the σ-1 receptor in HEK-293 cells stably expressing ASIC1a and transiently transfected with σ-1 receptor cDNA. Cells were incubated with mouse monoclonal anti-FLAG antibody, followed by fluorescein isothiocynate-conjugated goat anti-mouse secondary antibodies. A representative immunofluorescence image is shown, along with a corresponding brightfield image; ∼20% of the cells are expressing the σ-1 receptor. (C) Detection of ASIC1a in a membrane fraction from stably transfected cells (M) and after isolation (I) by binding to Ni²⁺-agarose. Samples were analyzed by SDS-PAGE and either silver staining (left panel) or immunoblotting (right panel) using rabbit polyclonal anti-ASIC1a antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). (D) Detection of ASIC1a and the σ-1 receptor after isolation from σ-1 receptor-transfected cells by binding to Ni²⁺-agarose. Samples were analyzed by SDS-PAGE and either silver staining (left panel) or immunoblotting (right panel) using anti-ASIC1a antibody or mouse monoclonal anti-FLAG antibody. (E) A screen for the presence of endogenous σ-1 receptor in protein samples isolated from nontransfected ASIC1a-expressing cells or σ-1 receptor-transfected cells. Samples were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody, or rabbit polyclonal anti-σ-1 receptor antibody.

Figure 2

AFM imaging of ASIC1a, anti-His₆ antibodies, and σ-1 receptors. (A) Low-magnification image of isolated ASIC1a. The arrow indicates a trimeric cluster of three equally sized particles, which is likely an ASIC trimer that has bound to the mica intact and then fallen apart during the drying process. A zoomed image of this cluster is shown at the bottom right of the panel. A shade-height scale is shown at the right. (B) Sections through the two particles indicated in A, taken at the positions indicated by the lines. The sections indicate that the shapes of the particles approximate that of a spherical cap. The calculated volumes of the particles are indicated. (C) Low-magnification image of anti-His₆ antibodies. A shade-height scale is shown at the right. (D) Frequency distribution of molecular volumes of anti-His₆ antibodies. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. (E) Low-magnification image of σ-1 receptors. A shade-height scale is shown at the right. (F) Frequency distribution of molecular volumes of σ-1 receptors. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.

Figure 3

Analysis of anti-His₆ antibody binding to His₈-tagged ASIC1a. (A) Low-magnification AFM images of a sample of isolated ASIC1a that had been incubated with anti-His₆ antibody. Arrowheads indicate singly decorated ASICs; arrows indicate doubly decorated ASICs. A shade-height scale is shown at the right. (B) Gallery of zoomed images of ASICs that are undecorated (top) or decorated by one (middle) or two (bottom) anti-His₆ antibodies. Lines indicate the angles between pairs of bound antibodies. (C–E) Frequency distributions of (C) molecular volumes of decorated central particles, (D) molecular volumes of bound peripheral particles, and (E) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

Figure 4

Analysis of σ-1 receptor binding to ASIC1a. (A) Gallery of zoomed images of ASICs that are undecorated (top) or decorated by one (middle) or two (bottom) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. (B–D) Frequency distributions of (B) molecular volumes of decorated central particles, (C) molecular volumes of bound peripheral particles, and (D) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

Figure 5

Decoration of bound σ-1 receptors with anti-FLAG antibodies. (A) Gallery of zoomed images of ASICs/σ-1 receptor complexes that are undecorated (top) or decorated by one (middle) or two (bottom) peripheral particles. Lines indicate the angles between pairs of bound large peripheral particles. (B) Frequency distribution of molecular volumes of bound peripheral particles. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. (C) Frequency distribution of angles between pairs of bound peripheral particles. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.

Figure 6

Determination of the raft association of ASIC1a and the σ-1 receptor. Cells were extracted with ice-cold Triton X-100 (1%) and subjected to centrifugation on Optiprep density gradients. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody (for the σ-1 receptor), or anti-caveolin antibody (as a raft marker).