Functional Expression in Escherichia coli of the Disulfide-Rich Sea Anemone Peptide APETx2, a Potent Blocker of Acid-Sensing Ion Channel 3

被引:25
作者
Anangi, Raveendra [1 ]
Rash, Lachlan D. [1 ]
Mobli, Mehdi [1 ]
King, Glenn F. [1 ]
机构
[1] Univ Queensland, Inst Mol Biosci, Brisbane, Qld 4072, Australia
来源
MARINE DRUGS | 2012年 / 10卷 / 07期
基金
英国医学研究理事会;
关键词
ASIC3; APETx2; heterologous expression; NMR; E; coli; SENSORY NEURONS; SECONDARY HYPERALGESIA; INFLAMMATORY PAIN; ASIC3; INHIBITOR; TARGET; PHARMACOLOGY; DISEASES; CURRENTS; STIMULI;
D O I
10.3390/md10071605
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
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 C-13/N-15-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.
引用
收藏
页码:1605 / 1618
页数:14
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