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
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.