Ligand and G-protein selectivity in the κ-opioid receptor.

The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.

Agonist-induced internalization of κ-opioid receptors in noradrenergic neurons of the rat locus coeruleus.

Kappa-opioid receptors (κOR) are positioned to modulate pre- and post-synaptic responses of norepinephrine-containing neurons in the rat locus coeruleus (LC). The ability of an acute systemic injection of a long acting κOR agonist, U50,488, to induce trafficking of κOR was assessed in the LC using immunogold-silver detection in male Sprague-Dawley rats. U50,488 administration shifted immunogold-silver labeling indicative of κOR from primarily plasmalemmal sites to intracellular sites when compared to vehicle-treated subjects. This translocation from the plasma membrane to the cytoplasmic compartment was prevented by pre-treatment with the κOR antagonist, norbinaltorphimine (norBNI). To determine whether agonist stimulation could induce adaptations in the expression of the noradrenergic synthesizing enzyme, dopamine beta hydroxylase (DßH), and κOR expression, Western blot analysis was used to compare expression levels of DßH and κOR following U50,488 administration. Expression levels for DßH and κOR were significantly increased following U50,488 administration when compared to controls. These data indicate that a systemic injection of a κOR agonist stimulates internalization of κORs in noradrenergic neurons and can impact κOR and DßH expression levels in this stress-sensitive brain region.

Subcellular targeting of kappa-opioid receptors in the rat nucleus locus coeruleus.

The dynorphin (DYN)-kappa opioid receptor (kappaOR) system has been implicated in stress modulation, depression, and relapse to drug-seeking behaviors. Previous anatomical and physiological data have indicated that the noradrenergic nucleus locus coeruleus (LC) is one site at which DYN may contribute to these effects. Using light microscopy, immunofluorescence, and electron microscopy, the present study investigated the cellular substrates for pre- and postsynaptic interactions of kappaOR in the LC. Dual immunocytochemical labeling for kappaOR and tyrosine hydroxylase (TH) or kappaOR and preprodynorphin (ppDYN) was examined in the same section of tissue. Light microscopic analysis revealed prominent kappaOR immunoreactivity in the nuclear core of the LC and in the peri-coerulear region where noradrenergic dendrites extend. Fluorescence and electron microscopy revealed kappaOR immunoreactivity within TH-immunoreactive somata and dendrites in the LC as well as localized to ppDYN-immunoreactive processes. In sections processed for kappaOR and TH, approximately 29% (200/688) of the kappaOR-containing axon terminals identified targeted TH-containing profiles. Approximately 49% (98/200) of the kappaOR-labeled axon terminals formed asymmetric synapses with TH-labeled dendrites. Sections processed for kappaOR and ppDYN showed that, of the axon terminals exhibiting kappaOR, 47% (223/477) also exhibited ppDYN. These findings indicate that kappaORs are poised to modulate LC activity by their localization to somata and dendrites. Furthermore, kappaORs are strategically localized to presynaptically modulate DYN afferent input to catecholamine-containing neurons in the LC. These data add to the growing literature showing that kappaORs can modulate diverse afferent signaling to the LC.