The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors

Citation
W. Xu et al., The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors, BIOCHEM, 39(45), 2000, pp. 13904-13915
Citations number
64
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
45
Year of publication
2000
Pages
13904 - 13915
Database
ISI
SICI code
0006-2960(20001114)39:45<13904:TCC7RI>2.0.ZU;2-H
Abstract
Binding pockets of the opioid receptors are presumably formed among the tra nsmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [H-3]diprenorph ine, an antagonist, to mu, delta, and kappa opioid receptors to charged met hanethiosulfonate (MTS) derivatives and identified the cysteine residues wi thin the TMDs that confer-red the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethy ltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [H-3]di prenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretre atment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [H-3]dipnnorphine binding with MTSEA a sensitivity in the order of kappa > mu much greater than delta. The effects of MTSEA occurred rapidl y, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-nal oxone, prevented the MTSEA effect, demonstrating that the reaction occurs w ithin or in the vicinity of the binding pockets. Each cysteine residue in t he TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [H-3]dipre norphine, and C7.38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA , respectively. These results indicate that the conserved Cys7.38 is differ entially accessible in the binding-site crevice of these receptors. The sec ond extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially acce ssible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine ac cessibility method.