Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

This study assessed how conformational information encoded by ligand binding to d-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gbg, and Kir3 subunits constitutively interacted with one another. GaoA associated with DORs and Gbg, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GaoA, DOR-Gbg, GaoA-Gbg, and GbgKir3 interfaces. Ligand-induced BRET changes assessing GbgKir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GaoA-Gbg interface, 2) displayed the same order of efficacy as those observed at the DOR-Gbg interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gbg/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gbg-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gbg with respect to DORs, GaoA, and channel subunits. Further, the fact that BRET changes at the Gbg-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.

Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile.

Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging. This study describes signaling diversity of mu opioid receptor (MOR) ligands in terms of logistic and operational parameters for ten different in vitro readouts. It then uses unsupervised clustering of curve parameters to: classify MOR ligands according to similarities in type and magnitude of response, associate resulting ligand categories with frequency of undesired events reported to the pharmacovigilance program of the Food and Drug Administration and associate signals to side effects. The ability of the classification method to associate specific in vitro signaling profiles to clinically relevant responses was corroborated using β2-adrenergic receptor ligands.

About 50 million of the U.S. adult population suffer from chronic pain. It is a complex disease in its own right for which currently available analgesics have been deemed woefully inadequate since ∼20% of the sufferers derive no benefit. Vitamin D, known for its role in calcium homeostasis and bone metabolism, is thought to be of clinical benefit in treating chronic pain without the side-effects of currently available analgesics. A strong correlation between hypovitaminosis D and incidence of bone pain is known. However, the potential underlying mechanisms by which vitamin D might exert its analgesic effects are poorly understood. In this review, we discuss pathways involved in pain sensing and processing primarily at the level of dorsal root ganglion (DRG) neurons and the potential interplay between vitamin D, its receptor (VDR) and known specific pain signaling pathways including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), epidermal growth factor receptor (EGFR), and opioid receptors. We also discuss how vitamin D/VDR might influence immune cells and pain sensitization as well as review the increasingly important topic of vitamin D toxicity. Further in vitro and in vivo experimental studies will be required to study these potential interactions specifically in pain models. Such studies could highlight the potential usefulness of vitamin D either alone or in combination with existing analgesics to better treat chronic pain.

Opiate drugs are the most effective analgesics available but their clinical use is restricted by severe side effects. Some of these undesired actions appear after repeated administration and are related to adaptive changes directed at counteracting the consequences of sustained opioid receptor activation. Here we will discuss adaptations that contribute to the development of tolerance. The focus of the first part of the review is set on molecular mechanisms involved in the regulation of opioid receptor signalling in heterologous expression systems and neurons. In the second part we assess how adaptations that take place in vivo may contribute to analgesic tolerance developed during repeated opioid administration.

Diabetic peripheral neuropathy (DPN) is the most incapacitating complication of diabetes mellitus. Up to 50% of patients with DPN develop peripheral neuropathic pain (PNP). The underlying ionic and molecular mechanisms of diabetic PNP (DPNP) are poorly understood. However, voltage gated potassium (K v 7) channels which have been implicated in the pathogenesis of other types of PNP are likely to be involved. Here we examined, in the streptozotocin (STZ) rat model of DPNP, whether activating the Kv7 channels with a potent activator retigabine (ezogabine) would reverse/attenuate behavioural signs of DPNP. STZ rats exhibited behavioural indices of mechanical and heat hypersensitivity, but not cold hypersensitivity or spontaneous pain, 35 days after STZ injection. Retigabine given at a dose of 15 mg/kg (but not at 7.5 mg/ kg, i.p.) significantly attenuated mechanical, but not heat hypersensitivity in DPNP rats, and was as effective as the positive control gabapentin. This analgesic effect of retigabine was completely reversed by the K v 7/M channel blocker XE991 (3 mg/kg, i.p.) indicating that the anti-allodynic effects of retigabine were mediated by K v 7 channels. In conclusion, the findings suggest that Kv7 channels are involved in DPNP pathogenesis, and that strategies that target their activation may prove to be effective in treating DPNP.

Kir3 channels control excitability in the nervous system and the heart. Their surface expression is strictly regulated, but mechanisms responsible for channel removal from the membrane remain incompletely understood. Using transfected cells, we show that Kir3.1/3.2 channels and delta opioid receptors (DORs) associate in a complex which persists during receptor activation, behaving as a scaffold that allows beta-arrestin (βarr) to interact with both signaling partners. This organization favored co-internalization of DORs and Kir3 channels in a βarr-dependent manner via a clathrin/dynamin-mediated endocytic path. Taken together, these findings identify a new way of modulating Kir3 channel availability at the membrane and assign a putatively novel role for βarrs in regulating canonical effectors for G protein-coupled receptors.