Abstract

The dermorphin-derived peptide [Dmt¹]DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂; Dmt, 2′,6′-dimethyltyrosine) labels μ-opioid receptors with high affinity and selectivity in receptor binding assays. In previous studies, [Dmt¹]DALDA displayed a mechanism of action distinct from that of morphine, as evidenced by its insensitivity to antisense probes reducing morphine analgesia and incomplete cross tolerance to morphine. In an effort to further elucidate the unusual mechanism of action, [³H][Dmt¹]DALDA has been synthesized and its binding profile studied. [³H][Dmt¹]DALDA binding was high affinity (K_D = 0.22 nM) and showed a regional distribution consistent with μ-receptors with highest levels in calf striatal membranes. [³H][Dmt¹]DALDA binding was far less sensitive than [³H][d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) to the effects of divalent and sodium cations and guanine nucleotides, although NaCl and guanosine 5′-(β,γ-imido)triphosphate together reduced specific [³H][Dmt¹]DALDA binding levels by almost 75%. Competition studies confirmed the μ-selectivity of the binding, with K_i values that were not appreciably different from those seen against [³H]DAMGO. In guanosine 5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding assays in brain and spinal cord membranes, [Dmt¹]DALDA was more potent than DAMGO, but showed plateaus suggestive of a partial agonist. [Dmt¹]DALDA bound to μ-opioid receptor clone 1 (MOR-1) and its splice variants with high affinity. Unlike [³H]DAMGO, [³H][Dmt¹]DALDA seemed to label both agonist and antagonist conformations of MOR-1 expressed in Chinese hamster ovary cells. In [³⁵S]GTPγS assays [Dmt¹]DALDA showed high efficacy with all the MOR-1 variants, but its potency (EC₅₀) varied markedly among some of the splice variants despite similar affinities in receptor binding assays. Although [³H][Dmt¹]DALDA is a very potent μ-selective analgesic, its binding characteristics and its ability to stimulate GTPγS binding differed from that of the classical μ-opioid peptide DAMGO.