Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up-or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is downregulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.

The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation-transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes Ca V 1.2 and Ca V 1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of Ca V 1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-Ca V 1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of Ca V 1.2 mRNA and protein in neuropathic animals concomitant to specific Ca V 1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on Ca V 1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising Ca V 1.2.

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA A receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X 3 or P2X 2+3 and GABA A receptors does not depend on chloride and calcium ions. We identified an intracellular QST [386][387][388] motif in P2X 3 subunits which is required for the functional coupling with GABA A receptors. Moreover the cross-inhibition between native P2X 3 and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA A b3 subunits. We provide evidence that P2X 3 and GABA A receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X 3 -GABA A subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.