Conformational Dynamics of Kir3.1/Kir3.2 Channel Activation Via δ-Opioid Receptors

Abstract

This study assessed how conformational information encoded by ligand binding to d-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gbg, and Kir3 subunits constitutively interacted with one another. GaoA associated with DORs and Gbg, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GaoA, DOR-Gbg, GaoA-Gbg, and GbgKir3 interfaces. Ligand-induced BRET changes assessing GbgKir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GaoA-Gbg interface, 2) displayed the same order of efficacy as those observed at the DOR-Gbg interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gbg/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gbg-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gbg with respect to DORs, GaoA, and channel subunits. Further, the fact that BRET changes at the Gbg-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.

Molecular Pharmacology ofδ-Opioid Receptors

“…BRET and coimmunoprecipitation assays revealed that DOPr may additionally associate with downstream effectors such as Kir3 channels (Richard-Lalonde et al, 2013;Nagi et al, 2015). The idea that GPCRs and Kir3 channel association may take place beyond overexpression systems is supported by biochemical and functional observations obtained with native dopamine D 2 and GABA B receptors in brain membranes.…”

“…Consensus similarly exists with respect to the constitutive association between heterotrimeric G proteins and Kir3 channel effectors (Bünemann et al, 2003;Riven et al, 2006;Robitaille et al, 2009;Berlin et al, 2010Berlin et al, , 2011Richard-Lalonde et al, 2013). Thus, both consensual observations imply that the receptor, G protein, and effector may be part of a single array containing all signaling partners even if for a limited time period, as has been described in HEK cells transfected with DOPrs, Gaob1g2 subunits, and Kir3.1/3.2 channels.…”

Endocytic Profiles ofδ-Opioid Receptor Ligands Determine the Duration of Rapid but Not Sustained cAMP Responses

“…The effect of agonists on DOR-G protein interaction was assessed using a method that we previously developed and validated for detection of ligand-induced conformational changes within DOR-Gprotein complexes (Gales et al, 2005(Gales et al, , 2006Audet et al, 2008Audet et al, , 2012Richard-Lalonde et al, 2013). Two days after transfection, HEK293 cells expressing the Gai1-Luc91/YFP-Gg 2 BRET pair plus accessory subunits and DORs were washed with PBS and distributed into 96-well plates at a concentration of 1 to 2 mg/ml.…”

“…Cells were then incubated for 3 minutes with coelanterazine H (1 mg/ml) before the addition of different ligands at increasing concentrations. BRET1 readings were taken 2 minutes after ligand introduction, and BRET ratios were corrected (net BRET) by subtracting the background signal detected when the RLuc-tagged construct was expressed without acceptor (Richard-Lalonde et al, 2013).…”

“…Opioid receptors belong to the subfamily of Gi/o-coupled receptors, and their stimulation results in canonical responses such as Kir3 channel activation, Cav2 channel inhibition, and reduction in cAMP production (McDowell, 2003;Audet et al, 2010;Nagi and Pineyro, 2011;Richard-Lalonde et al, 2013). All these responses are subject to regulation via a series of events that not only determine the duration of acute pharmacological responses but may also contribute to the development of analgesic tolerance (Williams et al, 2013).…”