两种Nav1.9专一性调制剂的作用机制研究和应用探索

Voltage-gated sodium channel subtype Nav1.9 plays an important role in pain signaling pathway and may be an idea target for the development of analgesic drugs. The pharmacological investigations and drug development based on this channel are therefore attracting great attention. Recently, scientists from basic study field and the pharmaceutical industry mainly focus on specific modulator discovery and action mechanism investigation. It should note that it is still hard for the heterologous expression of Nav1.9 in mammalian cells, which limits the progress of the discovery of Nav1.9 modulators. As a result, no specific modulators of Nav1.9 have been identified so far. In our previous study, we developed an effective Nav1.9 expression system through construction of chimeric channels. By using this system, we identified two high specific modulators of Nav1.9, named HTx1 and GTx23 through a large screening of peptide toxins. Patch clamp studies indicated that HTx1 delays the inactivation of Nav1.9, while GTx23 inhibits the activation of Nav1.9. Consequently, they demonstrated nociceptive and antinociceptive effects on mice, respectively. These data suggested both peptides might be potential pharmacological tools of Nav1.9 or drug leads. Subsequently, in the present study, we will try to elucidate the action mechanisms through investigating their effects on kinetics of Nav1.9 currents, construction of peptide-channel complex and determination of their nociceptive or antinociceptive behaviors, and therefore clarify the relationship between Nav1.9 function and pain. This study will expand our understanding for Nav1.9 regulating pain and provide basis for the analgesic drug development based on Nav1.9.

阐明钠通道Nav1.9参与疼痛信号通路调控是近年来国际疼痛研究领域的重要进展，其药理学研究和靶向Nav1.9研发镇痛新药因此成为研究热点，专一性调制剂筛选和作用机制研究是其中的重点内容。因缺少适于高通量筛选的Nav1.9哺乳动物细胞表达系统，其专一性调制剂未见报道，相关进展受阻。通过改造该通道C-端氨基酸序列构建了一种高效Nav1.9表达系统，并从本室储备的多肽毒素库中筛选和鉴定了两个高专一性Nav1.9调制剂HTx1和GTx23，分别对该通道电流有激活和抑制作用，且在小鼠模型表现为致痛和镇痛效应，是潜在的药理学工具试剂和药物先导分子。本项目以两个调制剂为研究对象，利用上述表达系统开展电生理活性、相互作用位点分析及应用探索，深入阐明其作用机制，在药理学层面，揭示Nav1.9电生理功能和疼痛的关系，扩展和深入Nav1.9疼痛相关研究，为靶向Nav1.9研发镇痛新药提供理论依据和先导分子。

钠通道Nav1.9参与疼痛信号通路调控是疼痛研究领域的重要内容，难以异源功能性表达的难题却阻碍了其电生理特性与生物学功能的解析，药理学工具的匮乏也制约了疼痛传导途径的深入探究。通过系统改造而来的Nav1.9异源表达系统则突破了该研究瓶颈为相关研究工作开拓道路，阐明了临床遗传突变体的发病机制以及炎症因子的联系，而高通量筛选鉴定到多种作用模式的多肽毒素调制剂，有效扩充了该通道的药理学工具试剂与药物先导分子。从白额高脚蛛毒液鉴定得到的HpTx1对疼痛密切相关的两种通道呈现相反作用模式，结合至Nav1.9 DIV s3b-s4区域抑制快失活而增强通道活性，但结合至Nav1.7 DII s3b-s4酸性氨基酸而抑制激活，系列作用机制研究则极力推动了通道结构功能关系研究，促进了靶向Nav1.9药物研发的设计。随后，具备独特调控模式的HpTx1应用至疼痛信号传导途径的探索，解析出HpTx1激活Nav1.9来增强神经元兴奋性达到恢复Nav1.7敲除小鼠疼痛不敏感表型的作用途径，阐述了Nav1.7、Nav1.8和Nav1.9分别在疼痛信号传导途径中的重要角色，填补了Nav1.9在疼痛信号传导研究中的药理学研究空白，也为先天性无痛症患者提供了药物先导分子和治疗新策略。

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