Functional expression in Escherichia coli of the disulfide-rich sea anemone peptide APETx2, a potent blocker of acid-sensing ion channel 3.
Functional expression in Escherichia coli of the disulfide-rich sea anemone peptide APETx2, a potent blocker of acid-sensing ion channel 3.
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DOI：
10.3390/md10071605
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发表时间：
2012-07
期刊：
影响因子：
5.4
通讯作者：
King GF
中科院分区：
文献类型：
--
作者：
Anangi R;Rash LD;Mobli M;King GF
酸感离子通道（asic）是存在于脊索动物中枢和外周神经系统的质子门控钠离子通道。ASIC3在感觉神经元中高度表达，在炎症性和缺血性疼痛中起重要作用。因此，ASIC3的特异性抑制剂具有开发新型镇痛药的潜力。APETx2是从海葵Anthopleura elegantissima中分离出来的，是最有效和选择性的asic3通道抑制剂。然而，APETx2的作用机制及其与ASIC3相互作用的分子基础尚不清楚。为了帮助表征ASIC3-APETx2的相互作用，我们开发了一种高效且具有成本效益的APETx2的大肠杆菌周质表达系统。对大肠杆菌中统一13C/ 15n标记的APETx2进行核磁共振研究表明，重组肽采用天然构象。重组APETx2与合成APETx2在抑制非洲爪蟾卵母细胞中ASIC3通道表达方面具有相同的能力。利用该系统，我们将Phe15突变为Ala，导致APETx2在ASIC3上的活性严重丧失。这些发现表明，该表达系统可用于产生APETx2的突变版本，以促进结构-活性关系的研究。
Acid-sensing ion channels (ASICs) are proton-gated sodium channels present in the central and peripheral nervous system of chordates. ASIC3 is highly expressed in sensory neurons and plays an important role in inflammatory and ischemic pain. Thus, specific inhibitors of ASIC3 have the potential to be developed as novel analgesics. APETx2, isolated from the sea anemone Anthopleura elegantissima, is the most potent and selective inhibitor of ASIC3-containing channels. However, the mechanism of action of APETx2 and the molecular basis for its interaction with ASIC3 is not known. In order to assist in characterizing the ASIC3-APETx2 interaction, we developed an efficient and cost-effective Escherichia coli periplasmic expression system for the production of APETx2. NMR studies on uniformly 13C/15N-labelled APETx2 produced in E. coli showed that the recombinant peptide adopts the native conformation. Recombinant APETx2 is equipotent with synthetic APETx2 at inhibiting ASIC3 channels expressed in Xenopus oocytes. Using this system we mutated Phe15 to Ala, which caused a profound loss of APETx2’s activity on ASIC3. These findings suggest that this expression system can be used to produce mutant versions of APETx2 in order to facilitate structure-activity relationship studies.
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