Wu Yangyu, Chen Zhuyuan, Sigworth Fred J, Canessa Cecilia M
Basic Sciences Department, Tsinghua University School of Medicine, Beijing, China.
Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, United States.
Elife. 2021 Jul 28;10:e67115. doi: 10.7554/eLife.67115.
ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.
ASIC1a是一种质子门控钠通道,参与疼痛、恐惧、成瘾和缺血诱导的神经元损伤的调节。我们报告了从羊驼中分离并鉴定出特异性靶向人ASIC1a的纳米抗体(Nbs)。在pH 7.4条件下,人ASIC1a通道与其中一种纳米抗体Nb.C1形成复合物的冷冻电子显微镜分析得到了分辨率为2.9 Å的结构。结果显示,Nb.C1结合的位点与德州珊瑚蛇毒素(MitTx1)和黑曼巴蛇毒Mambalgin-1的结合位点重叠;然而,Nb.C1的结合位点与抑制性狼蛛毒素Psalmotoxin-1(PcTx1)的结合位点不重叠。在单一多肽中将Nb.C1与PcTx1融合可显著增强PcTx1的效力,而Nb.C1和MitTx1竞争结合会降低毒素对通道的激活作用。因此,Nb.C1是用于hASIC1a生化和结构研究的分子工具;是珊瑚蛇咬伤致痛成分的潜在解毒剂;也是在体内增强PcTx1介导的对hASIC1a抑制作用以用于治疗应用的候选物。