Abstract
The G protein-gated inwardly rectifying K+ (GIRK) channels play important signaling roles in the central and peripheral nervous systems. However, the role of GIRK channel activation in pain signaling remains unknown mainly due to the lack of potent and selective GIRK channel activators until recently. The present study was designed to determine the effects and mechanisms of ML297, a selective GIRK1/2 activator, on nociception in the spinal cord by using behavioral studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons. Rats were prepared for chronic lumber catheterization and intrathecal administration of ML297. The nociceptive flexion reflex was tested using an analgesy-meter, and the influence on motor performance was assessed using an accelerating rotarod. We also investigated pre- and post-synaptic actions of ML297 in spinal cord preparations by whole-cell patch-clamp recordings. Intrathecal administration of ML297 increased the mechanical nociceptive threshold without impairing motor function. In voltage-clamp mode of patch-clamp recordings, bath application of ML297 induced outward currents in a dose-dependent manner. The ML297-induced currents demonstrated specific equilibrium potential like other families of potassium channels. At high concentration, ML297 depressed miniature excitatory postsynaptic currents (mEPSCs) but not their amplitude. The ML297-induced outward currents and suppression of mEPSCs were not inhibited by naloxone, a μ-opioid receptor antagonist. These results demonstrated that intrathecal ML297 showed the antinociceptive effect, which was mediated through direct activation of pre- and post-synaptic GIRK channels. Selective GIRK channel activation is a promising strategy for the development of new agents against chronic pain and opioid tolerance.
Highlights
The G protein-gated inwardly rectifying K+ (GIRK) channels are potassium-selective ion channels that act as effectors of Gi/o-coupled G protein-coupled receptors (GPCRs)
Since impaired motor function could explain the prolonged paw withdrawal latency, we examined the effect of intrathecal administration of ML297 on motor activity using the rotarod test
These results suggest that ML297 acts presynaptically, decreasing glutamate release, possibly due to the activity of GIRK channels
Summary
The G protein-gated inwardly rectifying K+ (GIRK) channels are potassium-selective ion channels that act as effectors of Gi/o-coupled G protein-coupled receptors (GPCRs). GPCRs such as opioid, adrenergic, muscarinic, and dopaminergic receptors, are involved in the regulation of pain transmission [4]. GIRK channels in the spinal cord are probably valuable therapeutic targets for pain management. It has been reported that ethanol directly activates several subtypes of GIRK channels without the involvement of G proteins or second messengers [5]. Ethanol exerts its effects on many channels, including NMDA and GABAA receptors, and it is not a specific GIRK activator. ML297 is a potent activator of the GIRK1/2 subunit combination having approximately 8-fold selectivity as compared to GIRK1/ 4 and has no activity towards a number of other potassium channels which show very limited expression in the brain [7]. Selective activation of GIRK channels by ML297 requires two amino acids specific to the GIRK subunit [8]. We determined first the antinociceptive effect of ML297 in rats using behavioral tests, used substantia gelatinosa (SG) neurons of spinal cord slices and wholecell patch-clamp technique to determine the cellular and molecular mechanisms of the antinociceptive effect of ML297
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