The role of a lysosomal mechano-sensitive ion channel in pain

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Project Summary/Abstract: Chronic pain reduces quality of life and work capability in millions of Americans. Currently, understanding of the underlying mechanisms and treatments for chronic pain are still limited. The long-term goal of this research is to identify major ion channel mechanisms and pharmacological targets for the treatment of neuropathic pain. The goal of the proposed research is to explore the role of a lysosomal ion channel (Tmem63A) in mediating functional currents in DRG neurons, and neuropathic pain associated with peripheral nerve injury. The central hypothesis of this proposal is that Tmem63A mediates a mechano-sensitive ion current in lysosomes of non- peptidergic nociceptive DRG neurons and positively contributes to neuropathic pain associated with peripheral nerve injury. Two specific aims are proposed in the current project: 1) Test that Tmem63A mediates a mechano-sensitive ion current in lysosomes, and facilitates lysosomal peripheral trafficking in non-peptidergic nociceptive DRG neurons; and 2) Test that functional up-regulation of Tmem63A contributes to neuropathic pain associated with peripheral nerve injury. In Aim 1, mouse DRG neurons will be dissociated. Dissociated DRG neurons will be subjected to virus-mediated knockdown or overexpression of Tmem63A. The cell type- specific, and subcellular-specific expression and mechano-sensitive currents of Tmem63A will be studied in four groups of DRG neurons using single-cell PCR, immunocytochemistry, and whole-lysosome patch clamp. Additional, lysosome positioning in these DRG neuron groups will also be studied by immunocytochemistry. In Aim 2, an in vivo repeated oxaliplatin treatment model (or chronic oxaliplatin treatment) of mouse will be used, and sensory behaviors (mechanical, heat and cold) will be assessed. A virus-mediated in vivo knockdown of Tmem63A in peripheral neurons will be conducted. The expression of Tmem63A will be detected in multiple sensory tissues using qPCR, Western blot, and immunohistochemistry methods. Lysosomal positioning will be examined by immunohistochemistry. The functional currents of Tmem63A will be measured in dissociated DRG neurons. The effects of oxaliplatin on expression and function of Tmem63A, lysosomal positioning will be studied. Moreover, the effects of Tmem63A knockdown on the oxaliplatin-induced neuropathic pain behaviors will be studied. To our knowledge, the roles of lysosomal-specific ion channels, or lysosomal mechano- sensitive ion channels in pain have not been reported. Therefore, the current study has the potential to establish lysosomal ion channels as potential targets for the treatment of multiple pain conditions.