Summary Opioids remain the standard for analgesic care, however adverse effects of systemic treatments contraindicate long-term administration. While most clinical opioids target mu opioid receptors (MOR), those that target the delta class (DOR) also demonstrate analgesic efficacy. Furthermore, peripherally-restrictive opioids represent an attractive direction for analgesia. However, opioid receptors including DOR are analgesically incompetent in the absence of inflammation. Here, we report that G Protein-Coupled Receptor Kinase 2 (GRK2) naively associates with plasma membrane DOR in peripheral sensory neurons to inhibit analgesic agonist efficacy. This interaction prevents optimal Gβ subunit association with the receptor, thereby reducing DOR activity. Importantly, bradykinin stimulates GRK2 movement away from DOR and onto Raf Kinase Inhibitory Protein (RKIP). Protein kinase C (PKC)-dependent RKIP phosphorylation induces GRK2 sequestration, restoring DOR functionality in sensory neurons. Together, these results expand the known function of GRK2, identifying a non-internalizing role to maintain peripheral DOR in an analgesically incompetent state.

Mechanical pain serves as a base clinical symptom for many of the world’s most debilitating syndromes. Ion channels expressed by peripheral sensory neurons largely contribute to mechanical hypersensitivity. Transient Receptor Potential A 1 (TRPA1) is a ligand-gated ion channel that contributes to inflammatory mechanical hypersensitivity, yet little is known as to the post-translational mechanism behind its somatosensitization. Here, we utilize biochemical, electrophysiological, and behavioral measures to demonstrate that metabotropic glutamate receptor-induced sensitization of TRPA1 nociceptors stimulates targeted modification of the receptor. Type 1 mGluR5 activation increases TRPA1 receptor agonist sensitivity in an AKA-dependent manner. As a scaffolding protein for Protein Kinases A and C (PKA and PKC, respectively), AKAP facilitates phosphorylation and sensitization of TRPA1 in ex vivo sensory neuronal preparations. Furthermore, hyperalgesic priming of mechanical hypersensitivity requires both TRPA1 and AKAP. Collectively, these results identify a novel AKAP-mediated biochemical mechanism that increases TRPA1 sensitivity in peripheral sensory neurons, and likely contributes to persistent mechanical hypersensitivity.

μ-Opioid receptor (MOR) agonists are often used to treat severe pain but can result in adverse side effects. To circumvent systemic side effects, targeting peripheral opioid receptors is an attractive alternative treatment for severe pain. Activation of the δ-opioid receptor (DOR) produces similar analgesia with reduced side effects. However, until primed by inflammation, peripheral DOR is analgesically incompetent, raising interest in the mechanism. We recently identified a novel role for G-protein-coupled receptor kinase 2 (GRK2) that renders DOR analgesically incompetent at the plasma membrane. However, the mechanism that maintains constitutive GRK2 association with DOR is unknown. Protein kinase A (PKA) phosphorylation of GRK2 at Ser-685 targets it to the plasma membrane. Protein kinase A-anchoring protein 79/150 (AKAP), residing at the plasma membrane in neurons, scaffolds PKA to target proteins to mediate downstream signal. Therefore, we sought to determine whether GRK2-mediated DOR desensitization is directed by PKA via AKAP scaffolding. Membrane fractions from cultured rat sensory neurons following AKAP siRNA transfection and from AKAP-knock-out mice had less PKA activity, GRK2 Ser-685 phosphorylation, and GRK2 plasma membrane targeting than controls. Site-directed mutagenesis revealed that GRK2 Ser-685 phosphorylation drives the association of GRK2 with plasma membrane-associated DOR. Moreover, overexpression studies with AKAP mutants indicated that impaired AKAP-mediated PKA scaffolding significantly reduces DOR-GRK2 association at the plasma membrane and consequently increases DOR activity in sensory neurons without a priming event. These findings suggest that AKAP scaffolds PKA to increase plasma membrane targeting and phosphorylation of GRK2 to maintain DOR analgesic incompetence in peripheral sensory neurons.

Introduction (426) Discussion (2062) d) Nonstandard abbreviations: dimethyl sulfoxide (DMSO), mean arterial pressure (MAP), opioid-induced respiratory depression (OIRD), phrenic nerve activity (PNA), vasopressin type 1a receptor (V1aR), vasopressin type 1a receptor antagonist (Phenylac 1 , D-Tyr(Et) 2 , Lys 6 , Arg 8 , des-Gly 9 )-Vasopressin trifluoroacetate (V1aRX) e) Recommended Section Assignment: Drug Discovery and Translational Medicine This article has not been copyedited and formatted. The final version may differ from this version.

Sleep apnoea increases susceptibility to opioid-induced respiratory depression (OIRD). r Endogenous opioids are implicated as a contributing factor in sleep apnoea. r Rats exposed to sleep-phase chronic intermittent hypercapnic hypoxia (CIHH) for 7 days exhibited exaggerated OIRD to systemic fentanyl both while anaesthetized and artificially ventilated and while conscious and breathing spontaneously, implicating heightened CNS inhibitory efficacy of fentanyl. r CIHH also induced tonic endogenous opioid suppression of neural inspiration. r Sleep-related episodes of hypercapnic hypoxia, as in sleep apnoea, promote hypersensitivity to OIRD, with tonic respiratory depression by endogenous opioids implicated as a potential underlying cause.