However, the single-site binding model had a very shallow nH of ?0.3 �� 0.1, and the data did not appear to fit this model well. The two-site binding model fits the data better (Figure?4B), giving pKi high 12.8 DAPT supplier
�� 0.6, n= 5 (Kihigh 175?fM; 37% of total binding) and pKi low 6.8 �� 0.2, n= 5 (Ki low 168?nM; 63% total binding). Using the extra sum of squares F-test, the two-site model was preferred with an F-value of 31.1 and P-value <0.0001. Due to the high affinity of IPAG for this high-affinity site, it is anticipated that depletion of the competing ligand (IPAG) had occurred. The affinity for this high-affinity site must therefore be considered an estimate. During these studies, we found that HEPES was a ligand at the sigma-1 receptor, with pKi 1.9 �� 0.5, n= 5 (Ki 12?mM), so therefore chose to buffer solutions using <a href="https://en.wikipedia.org/wiki/BML-190
">BML-190 Tris which was without effect on this receptor. In light of the observation that this competition curve resembles agonist competition curves binding to GPCRs (Itzhak, 1989; Connick et?al., 1992), we examined the effects of G protein-uncoupling agents on agonist and antagonist binding. Assays were performed in the presence and absence of GTP [1?mM, 10 times the normal physiological concentration of GTP (Reinhardt et?al., 2002)]. GTP was without effect on the binding of [3H]-(+)-pentazocine to the sigma-1 receptor in the permeabilized MDA-MB-468 cells; therefore, the competition assay with IPAG was repeated in the presence of GTP (Figure?5). The addition of GTP changed the shape of the binding curve, with a dramatic increase in the nH to ?0.81 �� 0.05. The affinity of IPAG for the sigma-1 receptor also shifts to the low-affinity site (pKi 6.3 �� 0.2, Ki 500?nM) in the presence of GTP, which has no significant difference from the low-affinity site seen this website
in the two-site fit in the absence of GTP (pKi 6.8; t-test P-value 0.183). Competition assays performed using intact cells also showed low-affinity binding to a single site [pKi 5.1 �� 0.2, n= 7 (Ki 8??M)]. We also tested a second sigma-1 receptor antagonist, rimcazole, which has a published affinity for this receptor of 0.9??M (Gilmore et?al., 2004) and has been previously shown to cause apoptosis in cancer cells through a sigma-1 receptor-dependent pathway (Spruce et?al., 2004). We were unable to use rimcazole in the calcium assay as it interfered with the wavelengths at which Fura-2 is read, fluorescing intensely at 510?nm. Indeed, we were able to produce a dose�Cresponse curve in the absence of Fura-2-loaded cells with an apparent EC50 of approximately 10??M (data not shown). However, rimcazole did not interfere with the reading of the MTS assay, and was found to inhibit metabolic activity of MDA-MB-468 cells with a pIC50 4.4 �� 0.2 (n= 5; IC50 45??M) which is over 30 times higher than the published affinity for the sigma-1 receptor (0.9??M; Gilmore et?al.