Modulation of Mu(1) opioid binding by magnesium: Evidence for multiple receptor conformations Journal Article
| Authors: | Standifer, K. M.; Clark, J. A.; Pasternak, G. W. |
| Article Title: | Modulation of Mu(1) opioid binding by magnesium: Evidence for multiple receptor conformations |
| Abstract: | 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 K(d) values with a significant increase in the B(max) 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 B(max) values of 1.89 ± 0.20 and 4.4 ± 0.19 fmol/mg wet weight tissue, respectively, with K(d) values of 3.8 ± 0.8 and 1.92 ± 0.27 nM. Despite the small but significant 2-fold difference in K(d) 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 (k(obs)) 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. First, [3H]DADLE rapidly binds to the receptor to form a high-affinity complex (L · R(h); τ( 1/2 ) 9 min), followed by a slower, MgSO4-dependent conversion of this receptor complex to a very high-affinity state with a far slower rate of dissociation (L · R(vh); τ( 1/2 ), 73 min). In the presence of MgSO4, L · R(vh) is formed directly from L · R(h) without dissociation of the ligand. EDTA prevents this conversion of L · R(h) to L · R(vh). |
| Keywords: | protein conformation; animal; metabolism; drug effect; kinetics; cattle; conformational transition; receptor affinity; mu opiate receptor; receptors, opioid, mu; magnesium sulfate; receptor binding; tritium; magnesium; dissociation constant; edetic acid; priority journal; article; enkephalin, leucine-2-alanine; support, u.s. gov't, p.h.s.; enkephalin, leucine 2 alanine; enkephalin[2 alanine 5 leucine] |
| Journal Title: | Journal of Pharmacology and Experimental Therapeutics |
| Volume: | 266 |
| Issue: | 1 |
| ISSN: | 0022-3565 |
| Publisher: | American Society for Pharmacology and Experimental Therapeutics |
| Date Published: | 1993-07-01 |
| Start Page: | 106 |
| End Page: | 113 |
| Language: | English |
| PUBMED: | 8392546 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 March 2019 -- Source: Scopus |
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