Upregulation of interleukin-6 on Ca(v)3.2 T-type calcium channels in dorsal root ganglion neurons contributes to neuropathic pain in rats with spinal nerve ligation.

The T-type calcium channels Cav3.2, one of the low voltage-activated (LVA) calcium channels, have been found to play important roles in the neuronal excitability. Recently, we and others have demonstrated that accumulation of Cav3.2 channels in the dorsal root ganglion (DRG) neurons and sensory nerves contributes to neuropathic pain after peripheral nerve injury. In the present study, we aimed to further investigate the regulation of Cav3.2 channels by interleukin-6 (IL-6) in DRG neurons in neuropathic pain rats after spinal nerve ligation (SNL). The results showed that Cav3.2 channel protein expression in L5 DRG neurons was upregulated and blockade of this channel decreased the hyperexcitability of DRG neurons and mechanical allodynia in SNL neuropathic pain rats. Furthermore, inhibition of IL-6 trans-signaling reduced the upregulation of Cav3.2 T-type channel induced by FIL-6 (a fusion protein of IL-6 and sIL-6R) in primary cultured DRG neurons in vitro. In vivo, inhibition of IL-6 trans-signaling reversed the upregulation of Cav3.2, reduced the hyperexcitability of L5 DRG neurons and alleviated mechanical allodynia in SNL rats. Our results suggest that IL-6 upregulates Cav3.2 T-type channels expression and function through the IL-6/sIL-6R trans-signaling pathway in DRG neurons, thus contributes to the development of neuropathic pain in SNL rats.

Liu Q ，Chen W ，Fan X ，Wang J ，Fu S ，Cui S ，Liao F ，Cai J ，Wang X ，Huang Y ，Su L ，Zhong L ，Yi M ，Liu F ，Wan Y ... -  《-》

Upregulation of Ca(v)3.2 T-type calcium channels in adjacent intact L4 dorsal root ganglion neurons in neuropathic pain rats with L5 spinal nerve ligation.

Besides the injured peripheral dorsal root ganglion (DRG) neurons, the adjacent intact DRG neurons also have important roles in neuropathic pain. Ion channels including Cav3.2 T-type calcium channel in the DRG neurons are important in the development of neuropathic pain. In the present study, we aimed to examine the expression of Cav3.2 T-type calcium channels in the intact DRG neurons in neuropathic pain. A neuropathic pain model of rat with lumbar 5 (L5) spinal nerve ligation (SNL) was established, in which the L4 DRG was separated from the axotomized L5 DRG, and the molecular, morphological and electrophysiological changes of Cav3.2 T-type calcium channels in L4 DRG neurons were investigated. Western blotting showed that total and membrane protein levels of Cav3.2 in L4 DRG neurons increased, and voltage-dependent patch clamp recordings revealed an increased T-type current density with a curve shift to the left in steady-state activation in the acutely isolated L4 DRG neurons in neuropathic pain rats. Immunofluorescent staining further showed that the membrane expression of Cav3.2 increased in CGRP-, IB4-positive small neurons and NF200-positive large ones. In conclusion, the membrane expression and the function of Cav3.2 T-type calcium channels are increased in the intact L4 DRG neurons in neuropathic pain rats with peripheral nerve injury like SNL.

Liu QY ，Chen W ，Cui S ，Liao FF ，Yi M ，Liu FY ，Wan Y ... -  《-》

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1-100 μM or ZC88, 10-1000 μM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 μM) and ZC88 (10 μM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1β) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1β induced mechanical allodynia and Cav2.2 upregulation in bilateral L4-6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1β and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1β over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

Yang J ，Xie MX ，Hu L ，Wang XF ，Mai JZ ，Li YY ，Wu N ，Zhang C ，Li J ，Pang RP ，Liu XG ... -  《-》

Cdk5-Dependent Phosphorylation of Ca(V)3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

Gomez K ，Calderón-Rivera A ，Sandoval A ，González-Ramírez R ，Vargas-Parada A ，Ojeda-Alonso J ，Granados-Soto V ，Delgado-Lezama R ，Felix R ... -  《-》

Increased expression of Ca(V)3.2 T-type calcium channels in damaged DRG neurons contributes to neuropathic pain in rats with spared nerve injury.

Kang XJ ，Chi YN ，Chen W ，Liu FY ，Cui S ，Liao FF ，Cai J ，Wan Y ... -  《Molecular Pain》