Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels
Isolation and structure-activity of μ-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels
复制标题
DOI：
10.1124/mol.106.028225
复制
发表时间：
2007-03-01
影响因子：
3.6
通讯作者：
Alewood, Paul F.
中科院分区：
文献类型：
--
作者：
Lewis, Richard J.;Schroeder, Christina I.;Alewood, Paul F.
芋螺毒素是锥螺在捕食过程中产生的抑制电压门控钠通道的三环多肽。利用聚合酶链式反应技术，从郁金香圆锥蛇毒导管中克隆了一种新的毒素TIIIA(TIIIA)的基因序列。建立了一种I-125-TIIIA结合分析方法，从斑腿螺粗毒中分离出天然的TIIIA。分离的多肽有三个翻译后修饰，包括两个羟基脯氨酸残基和C-末端的酰胺化，与其他魔芋毒素有35%的同源性。TIIIA能有效地从大鼠脑(Na(V)1.2)和骨骼肌(Na(V)1.4)膜上置换[H-3]TIIIA和~(125)I-TIIIA。TIIIA的丙氨酸和谷氨酰胺扫描显示，包括Arg14在内的几个残基对于高亲和力结合河豚毒素(TTX)敏感的Na+通道至关重要。我们惊讶地发现，[E15A]TIIIA对TTX敏感的钠通道的亲和力是TIIIA的10倍(大鼠脑膜IC50为15，而大鼠脑膜为148 PM)。TIIIA对非洲爪哇卵母细胞表达的Na(V)1.2和-1.4比Na(V)1.3、-1.5、-1.7和-1.8有选择性，对大鼠背根神经节神经元Na+电流无影响。H-1核磁共振研究表明，TIIIA在溶液中采用单一构象，该构象与之前所述的魔芋螺毒素PIIIA的主要构象相似。TIIIA及其类似物提供了新的生化探针以及对竹芋毒素结构活性的洞察。
mu-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A I-125-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and < 35% homology to other mu-conotoxins. TIIIA potently displaced [H-3] saxitoxin and 125I-TIIIA from rat brain (Na(v)1.2) and skeletal muscle (Na(v)1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Na(v)1.2 and -1.4 over Na(v)1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. H-1 NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.