Receptors Are Required for Full Inhibitory Coupling of Receptors to Voltage-Dependent Ca Channels in Dorsal Root Ganglion Neurons

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 channels (VDCCs) is a relatively inefficient process and therefore provides a sensitive assay of altered opioid receptor function and expression. We examined -receptor coupling to VDCCs in dorsal root ganglion neurons of ( / ), ( / ), and ( / ) mice. Neurons deficient in receptors exhibited reduced inhibition of VDCCs by morphine and [D-Ala,Phe,Gly-ol]-enkephalin (DAMGO). An absence of receptors caused reduced efficacy of DAMGO without affecting potency. An absence of receptors reduced neither the density of VDCCs nor their inhibition by either the GABAB receptor agonist baclofen or intracellular guanosine 5 -O-(3-thio)triphosphate. Flow cytometry revealed a reduction in -receptor surface expression in ( / ) neurons without altered DAMGO-induced internalization. There was no change in -receptor mRNA levels. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-PenThr-NH2-sensitive -receptor-coupling efficacy was fully restored to ( / ) levels in ( / ) neurons by expression of recombinant receptors. However, the dimerization-deficient -15 construct expressed in ( / ) neurons failed to fully restore the inhibitory coupling of receptors compared with that seen in ( / ) neurons, suggesting that, although not essential for -receptor function, receptor dimerization contributes to full -agonist efficacy. Because DAMGO exhibited a similar potency in ( / ) and ( / ) neurons and caused similar levels of internalization, the role for heterodimerization is probably at the level of receptor biosynthesis. Opioid receptors are the focus of intensive investigation because of their participation in nociception, mood, analgesia, and the rewarding effects of opioids and other abused substances. Opioid ( , , and ) receptors use common signaling mechanisms and have overlapping distributions in the central and peripheral nervous systems (Williams et al., 2001; Chen et al., 2008; Christie, 2008). However, despite these similarities, activation of different opioid receptor subtypes has distinctive behavioral consequences (Rees, 1992; Coop and Rice, 2000; Gavériaux-Ruff and Kieffer, 2002). The generation of -receptor knockout [ ( / )] mice led to the demonstration of the paramount importance of receptors in the analgesic and rewarding actions of morphine (Matthes et al., 1996). Systemic morphine administration had no discernible antinociceptive or rewarding effects in ( / ) mice. Furthermore, ( / ) mice lack the analgesic response to receptor agonists seen in wild-type animals, suggesting that much of this activity is mediated through partial activation of receptors (Scherrer et al., 2004). In contrast to ( / ) mice, receptor [ ( / )] and receptor [ ( / )] knockout mice exhibit intact morphine analgesia (Simonin et al., 1998; Scherrer et al., 2004). There is, however, evidence that receptors modulate receptor-mediated analgesia. Analgesia by intrathecal morphine is potentiated by a receptor antagonist (Gomes et al., 2004). Furthermore, ( / ) mice exhibit a reduced propensity for the developThis work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA05010, DA00484]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.055913. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: VDCC, voltage-dependent Ca channel; DAMGO, [D-Ala,Phe,Gly-ol]-enkephalin; DPDPE, [D-Pen,Pen]-enkephalin; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; GTP S, guanosine 5 -O-(3-thio)triphosphate; DRG, dorsal root ganglion; CFP, -cerulean fluorescent protein; PBS, phosphate-buffered saline; APC, allophycocyanin; FI, fluorescence intensity; CT, count threshold; HEK, human embryonic kidney. 0026-895X/09/7601-134–143$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 1 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 55913/3482960 Mol Pharmacol 76:134–143, 2009 Printed in U.S.A. 134 at U C LA B om ed Lib/S eria12-077 C tr or H lth S ci on A ril 3, 2014 m oharm .aspeurnals.org D ow nladed fom http://molpharm.aspetjournals.org/content/suppl/2009/04/08/mol.109.055913.DC1.html Supplemental Material can be found at: