Melatonin‐mediated inhibition of Cav3.2 T‐type Ca²⁺ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Abstract

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T-type Ca channels (T-type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T-type channels in small TG neurons via the melatonin receptor 2 (MT receptor) and a pertussis toxin-sensitive G-protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT receptor coprecipitated with Gα . Both shRNA-mediated knockdown of Gα and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin-induced T-type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC-eta (PKC ) while antagonism of PKC or shRNA targeting PKC prevented the melatonin-mediated effects. In a heterologous expression system, activation of MT receptor strongly inhibited Cav3.2 T-type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC dependent and was accompanied by a negative shift in the steady-state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain. These actions were inhibited by T-type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T-type channel activity through the MT receptor coupled to novel G -mediated PKC signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.

Melanocortin type 4 receptor–mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats

“…Electrophysiological procedures were performed at room temperature (23 Ϯ 1°C) as described previously (22,24). We chose only small-sized TG neurons that were less than 30 m in soma diameter for patch clamp experiments.…”

“…Western blot analyses were performed following a procedure described previously (22,24). In brief, 25 g of extracted proteins were separated in 7.5% SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Merck Millipore).…”

“…The standard procedure of immunohistochemistry was performed as described in our previous studies (22). Briefly, after sectioning (15 m) in a cryostat (CM 1950; Leica), TG sections were blocked with 5% normal goat serum in PBS plus 0.2% Triton X-100 for 1 h and then incubated overnight in the primary antibody against MC4R (rabbit, 1:500; Abcam), antibody against NF-200 (mouse, 1:1000; Abcam), or antibody against CGRP (mouse, 1:1000; Abcam).…”

“…Adenovirus-mediated gene silencing was performed following a procedure described previously (22,32). Three candidate shRNAs targeting p38␣ (GenBank TM accession number NM_031020.2) were designed, and the shRNA sequence (5Ј-GGACCTCCTTATAGACGAATG-3Ј) with the best knockdown efficiency were selected.…”

<p>Role of Melatonin in the Regulation of Pain</p>

“…This subsequently reduces neuronal excitability and ameliorates CFAinduced mechanical hypersensitivity. 111 Melatonin is able to suppress the mitogen-activated protein kinase (MAPK) and calcium signaling pathways via the MT2 receptor, which suppresses mechanical allodynia and thermal hyperalgesia induced by cuff-implanted. 17 The membrane receptors of melatonin are one of the most important mechanisms of its antinociception effect, especially MT2 receptor.…”