Differential regulation of the μ-, δ-, and κ-opiate receptor subtypes by guanyl nucleotides and metal ions

The effects of Na⁺, guanyl-5'-yl imidodiphosphate (Gpp(NH)p), and Mn²⁺ on the binding of dihydromorphine, ethylketocyclazocine, D-Ala², D-Leu⁵-enkephalin, and diprenorphine to the opiate receptor were investigated. Three distinct binding sites, μ, δ, and κ sites, were identified with the use of multiple tracer displacement curves. Moreover, this approach was used to determine the effects of Gpp(NH)p and metal ions on each individual binding site. At the μ and δ sites, Na⁺ and Gpp(NH)p each decreased and Mn²⁺ increased agonist binding affinities, with the exception of D-Ala², D-Leu⁵- enkephalin affinity which was not affected by Gpp(NH)p. None of these conditions markedly altered dihydromorphine and D-Ala², D-Leu⁵-enkephalin binding to κ sites, whereas the affinity of ethylketocyclazocine for κ sites was decreased by Gpp(NH)p. Sodium ions lowered the capacity of μ sites and Gpp(NH)p reduced that of δ sites, while both agents increased the capacity of apparent κ sites. These results demonstrate that each of the kinetically distinguishable binding sites is regulated differentially by metal ions and guanyl nucleotides. Simultaneous addition of Na⁺ and Gpp(NH)p greatly reduced the binding affinity of all three agonists at their respective high affinity sites (dihydromorphine at the μ site, D-Ala², D-Leu⁵-enkephalin at the δ site, and ethylketocyclazocine at the κ and μ sites). This result confirms previous observations that agonist binding is characterized by a large affinity reduction in the presence of both Na⁺ and guanyl nuleotides, and it extends this concept to each of the opiate receptor subtypes.