AGONIST INDUCED DESENSITIZATION OF MU-OPIOD RECEPTORS MEDIATED GIRK-CURRENTS.

Christophe Blanchet^1,2 and Christian Lüscher^1,2,3* Depts. of Physiology¹, Pharmacology (APSIC)², and Neurology³, Univ. of Geneva, CH 1211 Geneva, Switzerland.

Dependence and addiction to opiates are mediated by µ-opioid receptors (MORs). At the cellular level, MORs signal through the activation of G-protein coupled inwardly rectifying potassium (GIRK) channel, among other pathways. Here we have investigated the time-course of the GIRK current activated by MOR agonists in acute brain slices of the locus coeruleus (LC) of rats using standard whole-cell patch-clamp techniques at nearly physiological temperatures. An application of the selective MOR agonist DAMGO (1 m M) triggers an outward current of several hundreds of pA. Interestingly, this current desensitize to 67 ± 2% of its maximal initial amplitude within 15 minutes. As this acute desensitization may constitute a first step of neuroadaptation, we conducted a number of experiments to elucidate the underlying molecular mechanisms.
We found that the acute desensitization of MOR-mediated GIRK currents is concentration and agonist dependent. Since this correlates with the reported agonist dependent phosphorylation and internalization of MORs, we tested whether inhibition of kinases activity and/or disruption of receptor endocytosis affects desensitization. We found that, contrary to data from expression systems, neither manipulation affected desensitization of the MOR-induced GIRK currents. This dissociation suggests that MORs phosphorylation and internalization is a distinct step downstream of desensitization.
In cells of the LC, GIRK-currents may also be evoked by a a adrenergic receptor (a aAR) activation. These currents were subject to heterologous desensitization following MOR activation. Conversely, a aAR mediated-responses did not induce heterologous desensitization of MOR-mediated responses. These observations suggest a direct inhibitory effect of the MOR on GIRK channels, which are shared by both receptor systems. The special role of GIRK channels in desensitization is further corroborated by the observation of the absence of desensitization of MOR-induced presynaptic inhibition, an effect presumably mediated by Ca channel inhibition but not by GIRK channel activation. Desensitization of the GIRK-channel per se is however unlikely since we were able to restore maximal GIRK-current amplitude in the desensitized state by either photoreleased GTPg S or activation of a aARs by UK14304 (3 m M). Finally, while the photorelease of caged-GTPg S, through irreversible activation of G-protein of the Gi/o family, should activate GIRK current maximally and irreversibly, desensitization of the GIRK response still occured.
Taken together, our data suggest the involvement a novel G-protein dependent pathway, which would slowly inhibit GIRK currents after their rapid activation, thus leading to apparent desensitization. Our data may help to reveal targets for drugs, which would activate MORs, for example in pain treatment, without inducing dependence and addiction.