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There is ongoing debate about the role of G protein receptor kinases (GRKs) in agonist-induced desensitization of the μ opioid receptor (MOPr) in brain neurons (see Williams et al., 2013, Pharmacol Rev 15;223-254). In the present paper we have used a novel, membrane permeable, small molecule inhibitor of GRK2 and GRK3, Takeda Compound 101 (Cmpd101), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that Cmpd101 inhibits the desensitization of the G protein activated potassium current (GIRK) evoked by receptor- saturating concentrations of methionine enkephalin (Met Enk), D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO), endomorphin-2 and morphine in rat and mouse locus coeruleus (LC) neurons. In LC neurons from GRK3 knockout mice Met Enk-induced desensitization was unaffected implying a role for GRK2 in MOPr desensitization. Quantitative analysis of the loss of functional MOPrs following acute agonist exposure revealed that Cmpd101 only partially reversed MOPr desensitization. Inhibition of ERK1/2, PKC, JNK or GRK5 did not inhibit the Cmpd101-insensitive component of desensitization. In HEK 293 cells Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr phosphorylation at Ser375, arrestin translocation and MOPr internalization. Our data demonstrate a role for GRK2 (and potentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as yet unidentified, mechanism of desensitization also exists.