Antagonists show GTP-sensitive high-affinity binding to the sigma-1 receptor

Abstract

Background and purpose: Sigma-1 receptors are atypical receptors with potentially two transmembrane domains. Antagonists require doses significantly higher than their published affinities to have biological effects. We have reassessed the binding characteristics of these ligands and found antagonists bind to high- and low-affinity states not distinguished by agonists.

Experimental approach: The affinities of sigma-1 receptor ligands was assessed using radioligand saturation and competition binding of [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells. This was compared with the effect of ligands on metabolic activity using an MTS-based assay and calcium signalling using cells loaded with the calcium dye, Fura-2.

Key results: Sigma-1 receptor antagonists, but not agonists, show GTP- and suramin-sensitive high-affinity binding. Functional responses (calcium signalling and metabolic activity), while associated with sigma-1 receptor binding, required binding to an unidentified, low-affinity target.

Conclusions and implications: Sigma-1 receptors are coupled to G proteins. This interaction is only observed when analysing antagonist binding. The identity of the G protein remains to be resolved. The concept of agonist and antagonist at the sigma-1 receptor needs to be revisited.

Figure 1

Cytoplasmic [Ca²⁺] increase in response to IPAG. Calcium response to IPAG in Fura-2-AM-loaded MDA-MB-468 cells. Error bars show SEM, n = 6.

Figure 2

IPAG MTS assay dose–response. Cellular metabolic activity of MDA-MB-468 cells in response to IPAG, presented as a % of control. MTS was added 18 h after IPAG. Error bars show SEM, n = 5.

Figure 3

Effects of knocking down the sigma-1 receptor on cytoplasmic [Ca²⁺] response to IPAG. siRNA lowered maximal calcium response from 3100 ± 100 nM (non-targeting control) to 1600 ± 100 nM (mean ± SEM, n = 3). Error bars show SEM, n = 3. Parallel studies show receptor number was reduced from 1700 ± 100 fmol·mg⁻¹ protein (non-targeting control) to 800 ± 200 fmol·mg⁻¹ protein (mean ± SEM, n = 8).

Figure 4

IPAG binding to the sigma-1 receptor. IPAG competition binding curve with 30 nM [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells. (A) Using a curve which has been created using a single-site analysis. (B) Using a curve which has been created using a multiple-site analysis. Error bars show SEM, n = 5.

Figure 5

Effect of GTP (1 mM) on IPAG binding to the sigma-1 receptor. IPAG competition binding curve with 30 nM [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells washed with TBS to remove any endogenous GTP. Assays were performed in the absence (solid squares) or presence (open circles) of GTP. Competition binding to intact cells is also shown (solid diamonds). Error bars show SEM, n = 5.

Figure 6

Effect of GTP (1 mM) on rimcazole binding to the sigma-1 receptor. Rimcazole competition binding curve with 30 nM [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells washed with TBS to remove any endogenous GTP. Assays were performed in the absence (solid squares) or presence (open circles) of GTP. Error bars show SEM, n = 5.

Figure 7

Effect of suramin (100 µM) on IPAG binding to the sigma-1 receptor. IPAG competition binding curve with 30 nM [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells washed with TBS to remove any endogenous GTP. Assays were performed in the absence (solid squares) or presence (open circles) of suramin. Error bars show SEM, n = 5.

Figure 8

Effect of cholera toxin on IPAG-induced increase in cytoplasmic [Ca²⁺]. Representative (of n = 3) cytoplasmic Ca²⁺ response in Fura-2-loaded MDA-MB-468 cells following treatment with 100 µM IPAG. Solid line represents control cells; dashed line represents cells treated with 100 µg·mL⁻¹ cholera toxin overnight. IPAG was added at 50 s.