Tyrosine Phosphorylation of Kir3 following κ-Opioid Receptor Activation of p38 MAPK Causes Heterologous Desensitization

Abstract

Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K ir 3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K ir 3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wildtype mice, but not in mice lacking the -opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K ir 3.1-ir. The U50,488-induced increase in pY12-K ir 3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K ir 3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K ir 3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K ir 3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K ir 3.1-ir; and the heterologous desensitization of K ir 3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K ir 3.1 and channel inhibition through a GRK3-, p38 MAPK-and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.

Repeated Stress Dysregulates κ-Opioid Receptor Signaling in the Dorsal Raphe through a p38α MAPK-Dependent Mechanism

“…As it is typical for different rounds of staining to yield different immunoreactive intensities, images from each treatment group displayed in this study came from the same round of staining. The pattern of KORp-ir and GIRKp-ir across treatment groups seen in the dorsal raphe was similar to that previously reported both in vitro and in vitro (Ippolito et al, 2005; Bruchas et al, 2007; Xu et al, 2007; Land et al, 2008; Clayton et al, 2009). …”

Presynaptic Inhibition of Gamma-Aminobutyric Acid Release in the Bed Nucleus of the Stria Terminalis by Kappa Opioid Receptor Signaling

“…To determine if KOR signaling through these pathways was responsible for the reduction of GABA release in the BNST, we investigated the impact of a series of well-characterized pharmacological probes. To determine if p38MAPK signaling was required for KOR-mediated inhibition of GABA release, we bath applied the p38MAPK inhibitor (27), SB203580 (20 μM), for at least 45 minutes prior to beginning the experiment. This compound had no effect on the ability of 1 μM U69593 to inhibit eIPSCs in the BNST (66 ± 9 % of baseline, n = 6, Figure 6A, B).…”

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

“…There is less information regarding the role of initial part of the NT, which is missing in the crystal crystallography and NMR studies, in channel gating. Phosphorylation Tyr 12 of GIRK1 has been reported to regulate channel's I basal by unmasking a previously occluded GAP (GTPase-activating protein) domain that accelerates the GTPase activity of channel-associated Gα (Clayton, Xu, & Chavkin, 2009;Ippolito et al, 2002). Although direct biochemical support for a GAP activity of GIRK1 is lacking, this interesting hypothesis deserves further consideration and testing, with regard to the role and interactions of Gα with GIRK.…”