Cholecystokinin type B receptor-mediated inhibition of A-type K+ channels enhances sensory neuronal excitability through the phosphatidylinositol 3-kinase and c-Src-dependent JNK pathway

Abstract

Background Cholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown. Methods Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K + channels. Results CCK-8 reversibly and concentration-dependently decreased A-type K + channel ( I A ) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K + current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gα o . Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-G β antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated I A response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated I A decrease, whereas c-Src inhibition attenuated the CCK-8-induced p -JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by I A blockade. Conclusion Our findings indicate that CCK-8 attenuated I A through CCK-BR that is coupled to the G βγ -dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.

Single-chain Fragment variable antibody targeting cholecystokinin-B receptor for pain reduction

“…6 C, we observed a reduction in firing frequency in CCK-BR scFv-treated neurons compared to untreated neurons in the presence of CCK-8 (p < 0.0001, Mann-Whitney test). This concentration of CCK-8 used has previously been shown to sensitize sensory neurons in culture using electrophysiology assays ( Yu et al, 2019 ).…”

“…Previous work demonstrates that application of cholecystokinin, an endogenous agonist of CCK-B receptors, increases firing frequency of spinal dorsal root ganglia neurons through a decrease in A-type K + channel current (I A ) and caused mechanical and thermal hypersensitivity ( Yu et al, 2019 ). Indeed, our RNAseq studies indicate changes in several K + channel-related genes that would explain the effects of the scFv on nociceptor excitability, but requires further study.…”

The Cholecystokinin Type 2 Receptor, a Pharmacological Target for Pain Management

“…The underlying mechanism of CCK2R-mediated modulation of A-type K + currents involves the PI3K/SRC/JNK pathway. This signaling cascade results in the phosphorylation and, thus, the inhibition of A-type K + channels [48]. In the same study, the authors showed that intraplantar administration of CCK8 affected nociception in mice, characterized by mechanical and heat hypersensitivity, and that this effect was mediated by CCK2R and modulation of A-type currents.…”

“…Inhibition of the transient type-A K + current by CCK-8/CCK2R activation has been recently shown in small-sized DRG neuron extracts from mice [48]. DRG neurons express a variety of A-type K + channels that regulate membrane excitability.…”

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor

“…Evidence for the involvement of CCK2R in pain sensitization/perception is abundant. For example, CCK2R has been confirmed as being expressed in dorsal root ganglion neurons to mediate the inhibition of a Kv current (I A ) to increase pain sensation [16]. The CCK2R antagonist is known to reduce the need for opioid administration in relieving pain in a mouse model of burn-induced pain [17], likely by antagonizing CCK2R-µ-opioid receptor (MOR) heterodimerization, which inhibits MOR signaling [18].…”