δ Receptors Are Required for Full Inhibitory Coupling of μ Receptors to Voltage-Dependent Ca²⁺ Channels in Dorsal Root Ganglion Neurons

Abstract

Recombinant and ␦ opioid receptors expressed in cell lines can form heterodimers with distinctive properties and trafficking. However, a role for opioid receptor heterodimerization in neurons has yet to be identified. The inhibitory coupling of opioid receptors to voltage-dependent Ca 2ϩ channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. There was no change in -receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-PenThr-NH 2 -sensitive -receptor-coupling efficacy was fully restored to ␦(ϩ/ϩ) levels in ␦(Ϫ/Ϫ) neurons by expression of recombinant ␦ receptors. However, the dimerization-deficient ␦-15 construct expressed in ␦(Ϫ/Ϫ) neurons failed to fully restore the inhibitory coupling of receptors compared with that seen in ␦(ϩ/ϩ) neurons, suggesting that, although not essential for -receptor function, -␦ receptor dimerization contributes to full -agonist efficacy. Because DAMGO exhibited a similar potency in ␦(ϩ/ϩ) and ␦(Ϫ/Ϫ) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis.

Select G-Protein-Coupled Receptors Modulate Agonist-Induced Signaling via a ROCK, LIMK, and β-Arrestin 1 Pathway

“…After 1h of SNC80 (1μM), virally expressed cerulean-labeled δORs expressed in β-arr1+/+ and −/− neurons, demonstrate significant internalization (SFig 3). As δORs are dynamically trafficked to and from the cell membrane, the initial increase in δORs on the cell membrane could reflect delayed internalization and/or enhanced externalization in β-arr1−/− neurons (Cahill et al, 2007; Walwyn et al, 2009). …”

“…After 48h in vitro βarr1+/+ and −/− or δOR+/+ and −/− DRG neurons were harvested and processed for δOR fluorescent labeling in non-fixed cells using a primary antibody to the N-terminus of the δOR (MBL, Woburn, MA) and an Allophycocyanin (APC)-conjugated secondary antibody (BD Biosciences, San Jose, CA) as previously described (Walwyn et al, 2009). …”

“…D. A slower rate of internalization could enhance δOR-VDCC inhibition by increasing the number of δORs on the plasma membrane. This was assessed by over-expressing Cerulean (CFP)-labeled δORs (Walwyn et al, 2009). This increased δOR-VDCC inhibition equally in both β-arr1+/+ and−/− neurons tested with either DPDPE or SNC80 (1μM ea).…”

Modulation of Peripheral Sensory Neurons by the Immune System: Implications for Pain Therapy

“…The inhibition of Ca 2ϩ channels seems to be a major mechanism for the opioid-induced suppression of DRG neuron functions (Akins and McCleskey, 1993;Acosta and López, 1999;Khasabova et al, 2004;Wang et al, 2010). Interactions between different opioid receptor types, possibly via heterodimerization, can facilitate coupling to Ca 2ϩ channels (Walwyn et al, 2005(Walwyn et al, , 2009). Activation of peripheral opioid receptors also suppresses tetrodotoxin-resistant Na ϩ channels (Gold and Levine, 1996), nonselective cation currents (Ingram and Williams, 1994), purinergic 2X receptor-mediated currents (Chizhmakov et al, 2005), as well as TRPV1 currents via G i/o and the cAMP/protein kinase A pathway (Chizhmakov et al, 2005;Endres-Becker et al, 2007).…”

Preclinical Pharmacology of AZD2327: A Highly Selective Agonist of the δ-Opioid Receptor

“…The involvement of the opioid system in emotional regulation has been suggested for decades (Emrich et al, 1979;Tejedor-Real et al, 1995), although understanding of it is complicated by the multiplicity of receptor subtypes and endogenous ligands and potential hetero-oligomerization of the opioid receptors with each other as well as with other G protein-coupled receptors and ion channels (Walwyn et al, 2009). The three historically defined opioid subtypes are -opioid receptors, named after the prototypic agonist morphine; -opioid receptors, named after the benzomorphan opioid ketocyclazocine; and ␦-opioid receptors, named after observations of effects of agonists in a mouse vas deferens assay.…”