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.
Highlights
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that is involved in numerous cellular functions, such as cell migration and morphogenesis (Kupperman et al, 2000; Pyne and Pyne, 2000), lymphocyte egress (Pappu et al, 2007), angiogenesis and neurogenesis (Kono et al, 2004; Mizugishi et al, 2005)
To identify the S1P-activated chloride channel in dorsal root ganglia (DRG) neurons, we first searched for Ca2+-independent Cl− channels that are expressed in DRG based on our RNA sequencing data from wild-type mouse DRG explants
We selected CLCN3, CLCN4 and CLCN5 as possible candidates and investigated whether these three chloride channels contribute to S1P-activated chloride conductance in sensory neurons
Summary
Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that is involved in numerous cellular functions, such as cell migration and morphogenesis (Kupperman et al, 2000; Pyne and Pyne, 2000), lymphocyte egress (Pappu et al, 2007), angiogenesis and neurogenesis (Kono et al, 2004; Mizugishi et al, 2005). S1P is generated through conversion of ceramide into sphingosine by means of ceramidases and subsequent phosphorylation of sphingosine by sphingosine kinases (Pyne and Pyne, 2000; Spiegel and Milstien, 2003). S1P exerts its pleiotropic effects by signaling through a family of S1P receptors, consisting of five G-proteincoupled receptors designated S1PR1–5 (Spiegel and Milstien, 2003; Salvemini et al, 2013). S1PR1, S1PR4 and S1PR5 subtypes are mainly coupled to Gαi, whereas S1PR2 and S1PR3 subtypes are coupled to Gαi, Gq and Gα12/13 (Spiegel and Milstien, 2003; Brinkmann, 2007)
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