Two Distinct Mechanisms Mediate Acute μ-Opioid Receptor Desensitization in Native Neurons

Abstract

Sustained stimulation of G-protein coupled receptors (GPCRs) leads to rapid loss of receptor function (acute desensitization). For manyGPCRs including the -opioid receptor (MOR), an accepted mechanism for acute desensitization is through G-protein coupled receptor kinase (GRKs) mediated phosphorylation of the receptor, which facilitates the binding of ␤-arrestins (␤arrs) to the receptor and then promotes endocytosis. However, the mechanism(s) that mediate acute desensitization have not yet been well defined in native neurons. This study used whole-cell patch clamp recording of G-protein coupled inward-rectifying potassium (GIRK) currents to assay MOR function and identify mechanisms of acute MOR desensitization in locus ceruleus (LC) neurons. The rate and extent of MOR desensitization were unaffected by ␤arr-2 knock-out. Disruption of GRK2 function via inhibitory peptide introduced directly into neurons also failed to affect desensitization in wild type or ␤arr-2 knock-outs. Inhibition of ERK1/2 activation alone had little effect on acute desensitization. However, when both GRK2-␤arr-2 and ERK1/2 functions were disrupted simultaneously, desensitization of MOR was nearly abolished. Together, these results suggest that acute desensitization of MOR in native LC neurons is determined by at least two molecular pathways, one involving GRK2 and ␤arr-2, and a parallel pathway mediated by activated ERK1/2.

Cellular Morphine Tolerance Produced by βArrestin-2-Dependent Impairment of μ-Opioid Receptor Resensitization

“…Figure 1 confirms that acute desensitization of MOR-activated GIRK currents was unaffected by ␤arr-2 deletion (Dang et al, 2009). As reported previously in detail (Dang et al, 2009), the peak GIRK current activated by ME (30 M) was slightly smaller in ␤arr-2 k.o. than w.t.…”

“…mice and that GIRK activation can still be achieved by ␣2 adrenoceptor agonist, confirming that desensitization of MOR-activated GIRK is mostly homologous (Dang et al, 2009). Figure 1 confirms that acute desensitization of MOR-activated GIRK currents was unaffected by ␤arr-2 deletion (Dang et al, 2009). As reported previously in detail (Dang et al, 2009), the peak GIRK current activated by ME (30 M) was slightly smaller in ␤arr-2 k.o.…”

“…We previously reported that the rate and extent of agonist-induced acute desensitization of GIRK currents are nearly identical in LC neurons from w.t and ␤arr-2 k.o. mice and that GIRK activation can still be achieved by ␣2 adrenoceptor agonist, confirming that desensitization of MOR-activated GIRK is mostly homologous (Dang et al, 2009). Figure 1 confirms that acute desensitization of MOR-activated GIRK currents was unaffected by ␤arr-2 deletion (Dang et al, 2009).…”

“…Activation of MOR with a supramaximal concentration of the endogenous opioid ME (30 M, Ն10 min) has been widely reported to produce maximal desensitization and robust MOR endocytosis in LC neurons (Osborne and Williams, 1995;Bailey et al, 2004;Dang and Williams, 2005;Arttamangkul et al, 2006;Dang et al, 2009). Desensitization of MOR-activated GIRK currents in LC neurons provides a rapid, reliable assay of G-protein activation that does not involve loss of channel function because agonists at other GPCRs are still able to fully activate GIRK currents when MOR is desensitized in these cells (Connor et al, 2004).…”

“…mice may be caused by the loss of ␤arr-2-mediated MOR desensitization in neurons. However, studies in single locus ceruleus (LC) (Dang et al, 2009) and dorsal root ganglion (DRG) (Walwyn et al, 2007) neurons have shown that MOR desensitization is unaffected by ␤arr-2 deletion. Therefore, it remains unclear how ␤arr-2-dependent MOR regulation contributes to cellular opioid tolerance in native neurons.…”