Modulation of Mu1 opioid binding by magnesium: evidence for multiple receptor conformations

Previous studies have revealed that MgSO4 markedly increases mu1 binding and is associated with approximately a 10-fold slower dissociation rate, implying an increase in receptor affinity. Yet, saturation studies showed only a slight change in Kd values with a significant increase in the Bmax values. We now extend these observations using detailed kinetic approaches to better understand the role of MgSO4 on mu1 binding. Using blocking concentrations of the delta-selective ligand DPDPE, low concentrations of [3H]DADLE label mu1 sites almost exclusively in calf thalamic homogenates in the presence of either MgSO4 or EDTA, as demonstrated in competition studies. Saturation studies performed under equilibrium conditions with EDTA or MgSO4 reveal Bmax values of 1.89 +/- 0.20 and 4.4 +/- 0.19 fmol/mg wet weight tissue, respectively, with Kd values of 3.8 +/- 0.8 and 1.92 +/- 0.27 nM. Despite the small but significant 2-fold difference in Kd values under equilibrium conditions, kinetic studies reveal the dissociation rate constants (k-1) for the two assay conditions vary by almost 8-fold. NaCl increases the dissociation rate far more in assays performed in EDTA, while binding in the presence of MgSO4 is more sensitive to the stabilized GTP analog 5'-guanylylimidodiphosphate. Association studies also reveal a 20-fold difference between rate constants (k+1). The apparent association rate constants (kobs) in the presence of EDTA are concentration-dependent, whereas varying the concentration of [3H]DADLE has no effect on the rate constants under MgSO4 conditions. Our data suggest a model in which mu1 binding comprises two sequential steps and at least three distinct receptor binding states.(ABSTRACT TRUNCATED AT 250 WORDS)