Peripheral Kappa Opioid Receptor (KOR)‐Mediated Antinociception Requires G Protein‐Gated Inward Rectifying Potassium (GIRK) Channels

Abstract

Targeting peripherally expressed kappa opioid receptors (KOR) is a promising approach to treat inflammatory pain that would be devoid of CNS related adverse effects. KOR are known to couple to Gi‐protein‐mediated signaling pathways involved in the suppression of neuronal firing and nociception. In this study, we sought to determine the involvement of Gi protein coupled inward rectifying potassium (GIRK) channels in peripheral KOR‐mediated antinociceptive behavior in rats. Rats were injected intraplantarly (i.pl., 50ul) with PGE2 (0.3ug) with or without the KOR agonist, U50488 (0.1ug) and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. Treatment with U50488 completely reduced the thermal allodynia produced by PGE2. Consistent with activation of Gi proteins by KOR, we found that the reduction of PGE2‐evoked thermal allodynia by U50488 was lost completely 24h after i.pl. injection of pertussis toxin. To determine if GIRK channels mediated the antinociceptive response to KOR, we measured the effect of U50488 on PGE2‐evoked thermal allodynia 15 minutes after i.pl. injection of either vehicle or tertiapin Q (TPNQ), an inhibitor that blocks channels containing GIRK1 and GIRK2 subunits. Administration of TPNQ had no effect on either baseline PWL or PGE2‐evoked thermal allodynia, however, U50488‐mediated reduction of PGE2‐evoked thermal allodynia was lost after TPNQ administration. We next tested if direct activation of GIRK channels by i.pl. injection of the GIRK activator, ML297, could produce antinociceptive effects. ML297 (33ug) produced a robust analgesic effect (+5 sec above baseline) compared to vehicle treated animals as well as prevented PGE2‐evoked thermal allodynia. To further corroborate the importance of GIRK channels in peripheral KOR mediated antinociception, we tested effects of siRNA‐mediated knockdown of GIRK2 channel subunits following local administration of GIRK2 Accell‐siRNA (Dharmacon) into the hindpaw. After two daily i.pl. injections of GIRK2 siRNA, neither baseline PWL nor PGE2‐evoked thermal allodynia were altered, however, U50488‐mediated reduction in PGE2‐evoked thermal allodynia was lost. We verified knockdown of the GIRK2 subunit in hindpaws by RT‐qPCR and immunohistochemistry. Compared to the contralateral (CONTRA) hindpaw, GIRK2 mRNA was decreased significantly in the ipsilateral (IPSI) hindpaw. Further, there was no difference between IPSI and CONTRA hindpaws in the immunofluorescence for PGP9.5, a marker used to identify neuronal processes, or DAPI, a cell nuclei marker. By contrast, GIRK2 immunofluorescence was significantly decreased in IPSI hindpaws compared to CONTRA hindpaws suggesting that the expression of GIRK2 was indeed reduced. Together these results suggest that peripherally mediated antinociception in response to activation of KOR is due, in large part, to activation of GIRK channels.Support or Funding InformationThis work is supported by NIH/NIDA RO1 DA 038645 and NIH/NIGMS RO1 GM 106035.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.