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Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels

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Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels

Elsa Cortés-Montero et al. Front Pharmacol.

Abstract

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R-NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R-binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R-NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R-BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.

Keywords: N-methyl-D-aspartate receptor; calmodulin; histidine triad nucleotide-binding protein 1; ligand bias; transient receptor potential ankyrin member 1; transient receptor potential melastatin member 8; transient receptor potential vanilloid member 1; type 1 sigma receptor.

Figures

Figure 1
Figure 1
Sigma 1 receptor (σ1R), histidine triad nucleotide-binding protein 1 (HINT1), and calmodulin (CaM) binding to the transient receptor potential ankyrin member 1 (TRPA1) calcium channel. The TRP structural models shown were predicted by Novafold (DNASTAR Inc., Madison, WI, USA). Linear model: the N- and C-terminal cytosolic sequences are red and green, respectively, and the six transmembrane domains are in gray. Ribbon model: The 3D structure of N- and C-terminal sequences is shown; the CaM-binding motifs are indicated by blue spheres. (A) The in vitro interactions of the σ1R, CaM, and HINT1 with TRPA1 were evaluated in co-precipitation assays. Recombinant N- and C-terminal regions of TRPA1 (100 nM) were co-incubated in the presence and absence of 3 mM CaCl2, with the input of 200 nM CaM, σ1R, and HINT1. The TRPA1 N-terminus (aa 1–721) or the TRPA1 C-terminus (aa 961–1125) were immobilized by covalent attachment to NHS-activated Sepharose. Prey proteins alone did not bind to the blocked NHS-Sepharose (negative control). (B and C) Competition assays between the σ1R and CaM or HINT1 for binding to the N- and C-terminal regions of TRPA1. After incubation in 3 mM CaCl2, the TRPA1-bound proteins were detached and resolved by SDS-PAGE chromatography, and analyzed in Western blots. The assays were repeated at least twice, producing comparable results. For the interactions with increased concentrations of the σ1R (up to 200 nM), the data are shown relative to that obtained in the absence of the σ1R, with the control group arbitrary assigned a value of 1. *Significant differences with respect to the control group, ANOVA and Dunnett multiple comparisons vs. control group, p < 0.05. Representative blots are shown.
Figure 2
Figure 2
σ1R, HINT1, and CaM binding to the transient receptor potential melastatin member 8 (TRPM8) calcium channel. (A) Interactions between the σ1R, CaM, and HINT1 with the TRPM8 N-terminus (aa 1–639). Recombinant TRPM8 N-terminus (100 nM) was incubated with CaM, σ1R, and HINT1 (200 nM) in the presence or absence of 3 mM CaCl2. (B) Competition experiments to evaluate the interference of σ1R binding with that of CaM and HINT1 to the TRPM8 N-terminus (details as in Figure 1 ).
Figure 3
Figure 3
σ1R, HINT1, and CaM binding to the transient receptor potential vanilloid member 1 (TRPV1) calcium channel. (A) Interactions between σ1R, CaM, and HINT1 with the TRPV1. The recombinant TRPV1 N-terminus (aa 1–433) and C-terminus (aa 680–839) (100 nM) were incubated with CaM, σ1R, and HINT1 (200 nM) in the presence or absence of 3 mM CaCl2. (B and C) Competition assays between the σ1R and CaM or HINT1 for their binding to the N- and C-terminal regions of TRPV1 (details as in Figure 1 ).
Figure 4
Figure 4
The effect of σ1R ligands on the σ1R–TRP interactions. Agarose-TRP was incubated with the σ1R and the agarose–TRP–σ1R complexes were separated from the free σ1R through three cycles of washing/resuspension. The agarose–TRP–σ1R complexes were then incubated for 30 min with rotation at room temperature (RT) in the presence of increasing concentrations of the σ1R ligands in a final volume of 300 µL (50 mM Tris–HCl [pH 7.5], 3 mM CaCl2, and 0.2% CHAPS). Finally, the σ1Rs that remained attached to the TRP were resolved by SDS-PAGE and evaluated in immunoblots. Agonists dampened on the association of the σ1Rs with TRPA1, while antagonist induced different effects on σ1Rs binding to the TRPA1 N- or C-terminal domains. The σ1R agonists produced different effects on the TRPV1-σ1R associations, and while the antagonists did not alter these associations at TRPV1 N-terminus, they did diminish these complexes at the TRPV1 C-terminal domain. The association of TRPM8 with the σ1R was enhanced by agonists and dampened by antagonists. The assays were performed twice, and each point was duplicated. Representative blots are shown. PregS, pregnenolone sulfate; ProG, progesterone. Details as in Figure 1 .
Figure 5
Figure 5
The effect of agonism/antagonism of σ1R ligands on σ1R–TRP interactions. Agarose TRP–σ1R complexes were incubated in the presence of the competing σ1R ligands as indicated. The σ1Rs that remained attached to the TRP were evaluated in immunoblots: *Significant differences with respect to the control group; ϕsignificant difference with respect to the group receiving only the ligand that diminished the σ1R–TRP interaction. ANOVA and Dunnett multiple comparisons vs. control group, p < 0.05. Details as in Figures 1 and 4 .

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