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

James Brimson1, Colin Brown2, Stephen Safrany2,1. 1University of Bath, Department of Pharmacy and Pharmacology, United Kingdom, 2University of Wolverhampton, Department of Pharmacy, United Kingdom.

Background Sigma-1 receptors are atypical cell surface receptors with potentially two transmembrane domains. Antagonists at these receptors are potential anticancer drugs, whereas agonists show antidepressant activity. 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.

Methods The affinities of sigma-1 receptor ligands was assessed using radioligand saturation and competition binding of [3H](+) pentazocine to permeabilised 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.

Results Binding of the agonist (+) pentazocine was simple with pKd ± SEM of [3H] 7.8 ± 0.6, Kd 17 nM (n = 3). GTP (1mM) did not affect binding characteristics. 1-(4-iodophenyl)-3-(2-adamantyl)guanidine (IPAG) competition assays showed IPAG bound two affinity states, with pKi high 12.8 ± 0.6, n = 5 (Ki high 175 fM) (37% of total binding) and pKi low 6.8 ± 0.2, n = 5 (Ki low 168 nM) (63% total binding) (n = 5). The addition of GTP (1mM) caused a loss of high affinity binding of IPAG with simple binding to the low affinity site observed (pKi 6.3 ± 0.2, Ki 50 nM) (n = 5). The pEC50 for IPAG in the calcium response assay was 3.9 ± 0.1, (EC50 123 μM) (n = 6). IPAG also dose-dependently led to decreased cellular proliferation. The pIC50 for IPAG in this assay was 4.6 ± 0.1, (IC50 24 μM) (n = 5). Both these responses lie to the right of receptor occupancy, suggesting IPAG’s effects are mediated via a different target. However, SiRNA-mediated knock-down the sigma-1 receptor by approximately 50% resulted in a decrease in the reduction in maximal Ca2+ response by 50%, but did not affect the EC50 identified that the sigma-1 receptor is a functional target involved in the cellular response to IPAG.

Conclusions Sigma-1 receptors are coupled to an unidentified G-protein. This interaction is only observed when analysing antagonist binding. The concept of agonist and antagonist at the sigma-1 receptor needs to be revisited. A second target for IPAG be it on the sigma-1 receptor or elsewhere needs to be identified.