Institute of Physiology (M.R., A.O.-B., S.G., A.S.) and Department of Oncology, Hematology and Stem Cell Transplantation (G.R.), RWTH Aachen University, Aachen, Germany; and Computational Biomedicine - Institute for Advanced Simulation (IAS)/Institute of Neuroscience and Medicine (INM) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, Jülich, Germany (M.A.M., G.R.).
Institute of Physiology (M.R., A.O.-B., S.G., A.S.) and Department of Oncology, Hematology and Stem Cell Transplantation (G.R.), RWTH Aachen University, Aachen, Germany; and Computational Biomedicine - Institute for Advanced Simulation (IAS)/Institute of Neuroscience and Medicine (INM) and Jülich Supercomputing Centre (JSC), Forschungszentrum Jülich, Jülich, Germany (M.A.M., G.R.)
Mol Pharmacol. 2018 Oct;94(4):1114-1124. doi: 10.1124/mol.118.112375. Epub 2018 Jul 16.
Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na channel with important roles in pain. ASIC3 quickly desensitizes in less than a second, limiting its capacity to sense sustained acidosis during pain. RFamide neuropeptides are modulators of ASIC3 that slow its desensitization and induce a variable sustained current. The molecular mechanism of slowed desensitization and the RFamide binding site on ASIC3 are unknown. RPRFamide, a RFamide from the venom of a cone snail, has a comparatively high affinity for ASIC3 and strongly slows its desensitization. Here we show that covalent binding of a UV-sensitive RPRFamide variant to ASIC3 prevents desensitization, suggesting that RPRFamide has to unbind from ASIC3 before it can desensitize. Moreover, we show by in silico docking to a homology model of ASIC3 that a cavity in the lower palm domain, which is also known as the nonproton ligand-sensing domain, is a potential binding site of RPRFamide. Finally, using extensive mutagenesis of residues lining the nonproton ligand-sensing domain, we confirm that this domain is essential for RPRFamide modulation of ASIC3. As comparative analysis of ASIC crystal structures in the open and in the desensitized conformation suggests that the lower palm domain contracts during desensitization, our results collectively suggest that RPRFamide, and probably also other RFamide neuropeptides, bind to the nonproton ligand-sensing domain to stabilize the open conformation of ASIC3.
酸敏离子通道 3（ASIC3）是一种质子门控 Na+通道，在疼痛中具有重要作用。ASIC3 在不到一秒的时间内快速脱敏，限制了其在疼痛过程中感知持续酸中毒的能力。RFamide 神经肽是 ASIC3 的调节剂，可减缓其脱敏作用并诱导可变的持续电流。减缓脱敏的分子机制和 ASIC3 上的 RFamide 结合位点尚不清楚。RPRFamide 是一种来自 cone snail 毒液的 RFamide，对 ASIC3 具有相对较高的亲和力，并强烈减缓其脱敏作用。在这里，我们表明，与 ASIC3 共价结合的 UV 敏感 RPRFamide 变体可防止脱敏，表明 RPRFamide 必须从 ASIC3 上解离后才能脱敏。此外，我们通过同源 ASIC3 模型的计算机对接表明，下手掌域（也称为非质子配体感应域）中的一个腔是 RPRFamide 的潜在结合位点。最后，通过对非质子配体感应域周围残基进行广泛的诱变，我们证实该域对于 RPRFamide 调节 ASIC3 是必不可少的。由于对开放和脱敏构象中的 ASIC 晶体结构的比较分析表明，下手掌域在脱敏过程中收缩，我们的结果共同表明，RPRFamide 可能还有其他 RFamide 神经肽，与非质子配体感应域结合以稳定 ASIC3 的开放构象。
