Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Targeting the Sphingosine-1-Phosphate Axis for Developing Non-narcotic Pain Therapeutics

“…While the downstream signaling events are not yet entirely understood, the proposed mechanism involves S1PR3-Gα13-dependent regulation of RhoA (Figure 3). Since phosphorylation is one regulatory mechanism that controls chloride channel activity [70] and RhoA can modulate p38-MAPK activity [72], p38-MAPK signaling may regulate S1P-induced Clefflux in DRG neurons [71]. However, S1P-induced Clcurrents and potentiation of TRPV1 functions mediated by S1P in sensory neurons described earlier seem to be independent molecular mechanisms [71].…”

“…Nociceptive inputs are then sent to the cell bodies in the DRG and relayed to the spinal cord and brain to be processed [69]. Besides the aforementioned sodium and calcium currents, in DRG sensory neurons, chloride currents have also been implicated in the transmission of peripheral nociceptive stimulation [70], where two voltage-gated chloride channels (CLCN), CLCN3 and CLCN5, directly regulate excitatory currents triggered by S1P stimulation [71]. While the downstream signaling events are not yet entirely understood, the proposed mechanism involves S1PR3-Gα13-dependent regulation of RhoA (Figure 3).…”

“…However, several therapeutic agents that lack activity at S1PR2 (i.e., FTY720) show beneficial results in models of neuropathic pain states, suggesting a noncritical involvement of this receptor [84,98,99]. Emerging evidence also points to interesting contributions of S1PR3 [36,61,71] and development of highly selective S1PR3 antagonists could provide valuable tools for in-depth exploration of their effects for pain treatment. There is no doubt that the S1P/S1PR1 axis represents a fertile area for therapeutic intervention.…”

“…All other authors declare no conflict of interest. [36] CLCN3 and CLCN5 mediate excitatory Clcurrents activated by S1P/S1PR3 in mouse sensory neurons 2018 [71] S1P-to-S1PR3 signaling mediates pain behaviors in mice through the activation of TRPV1 in sensory neurons 2018 [61] Sexual dysmorphic responses are observed with bortezomib. Unlike paclitaxel and oxaliplatin, S1PR1-based therapies do not block bortezomib-induced neuropathic pain in female rats and mice 2019 [111] Ceramide-induced pain behaviors in mice involve NLRP2 inflammasome activation in the DRG 2019 [50] Activation of S1PR1 in spinal astrocytes, following intrathecal injection of SEW2871, leads to NLRP3 inflammasome activation, IL-1β production, and pain behaviors in mice and rats 2019 [81] Functional and competitive S1PR1 antagonists block traumatic nerve injury-induced neuropathic pain in mice and rats.…”

Chloride – The Underrated Ion in Nociceptors

“…As a consequence of the high intracellular Cl − concentration mediated by the active accumulation of Cl − by NKCC1, for example GABA-evoked depolarizing currents are observed in primary afferents at resting membrane potential (Sung et al, 2000); likewise, activation of G protein-coupled receptors, e.g. by lysophosphatidic acid (LPA) and sphingosine-1phosphate (S1P), has been shown to activate excitatory chloride conductances (Ponsioen et al, 2009;Camprubi-Robles et al, 2013;Qi et al, 2018).…”