Two critical residues in p-loop regions of puffer fish Na+ channels on TTX sensitivity
Two critical residues in p-loop regions of puffer fish Na+ channels on TTX sensitivity
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DOI：
10.1016/j.toxicon.2007.10.014
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发表时间：
2008-03-01
期刊：
影响因子：
2.8
通讯作者：
Yotsu-Yamashita, Mari
中科院分区：
文献类型：
--
作者：
Maruta, Satoshi;Yamaoka, Kaoru;Yotsu-Yamashita, Mari
我们之前表明，河豚鱼、东方鲀、骨骼肌Na-v（fNa（v）1.4a）的结构域I和IV中p环区域的Asn-383和Thr-1569残基分别与其他物种的TTX敏感性Na+通道不同，其中Phe或Tyr和Gly的芳香残基是对应物[Yotsu-Yamashita，M.，西森，K.，Nitanai，Y.，Isemura，M.，杉本，A.，Yasumoto，T.，2000. ~ 3 H-PbTx-3和~ 3 H-saxitoxin与河豚（Fugu pardatis）脑膜和骨骼肌膜的结合特性及河豚骨骼肌电压门控Na ~+通道α亚基（fMNa 1）的一级结构生物化学、生物物理通信资源267，403-412]。建议通过使用rNa（v）1.4的Y 401 N突变体来赋予TTX抗性[Venkatesh，V.，Lu，S.Q.，Dandona，N.，你看S. L Benner，S.，宋子文，2005.河豚鱼对河豚毒素抗性的遗传基础。Curr. 15，2069-2072]。后一项职能仍有待阐明。因此，我们进一步探索了这两个残基的功能，电生理，通过评估K-d在HEK-293细胞中瞬时表达的rNa（v）1.2a的F385 N、F385 A、F385 Q、G1718 T和F385 N/GI 718 T突变体的TTX（解离常数）值F385 N导致K-d增加3000倍，而G1718 T和F385 N/GI 718 T分别比WT和F385 N增加了2倍和3倍，表明G1718 T进一步增强了F385 N引起的TTX电阻率。F385 A和F385 Q的Kd分别是F385 N的2倍和11倍，表明非芳香族氨基酸残基中的较长侧链导致TTX敏感性的较大降低。尽管K-d发生了剧烈变化，但F385处的突变仅引起了WT的k（off）的微小变化，这表明TTX受体的Kd主要由k（on）决定。(c)2007爱思唯尔有限公司保留所有权利。
We previously showed that Asn-383 and Thr-1569 residues of p-loop regions in domains I and IV, respectively, of the puffer fish, Fugu pardialis, skeletal muscle Na-v (fNa(v)1.4a), are anomalous to those of other species of TTX-sensitive Na+ channels, where the aromatic residues of Phe or Tyr, and Gly are the counterparts [Yotsu-Yamashita, M., Nishimori, K., Nitanai, Y., Isemura, M., Sugimoto, A., Yasumoto, T., 2000. Binding properties of 3 H-PbTx-3 and 3 H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardatis and the primary structure of a voltage-gated Na+ channel a-subunit (fMNa 1) from skeletal muscle of F pardalis. Biochem. Biophys. Res. Commun. 267, 403-412]. The ormer was suggested to confer TTX resistance by using Y401N mutant of rNa(v)1.4 [Venkatesh, V., Lu, S.Q., Dandona, N., See, S.L., Benner, S., Soong, T.W., 2005. Genetic basis of tetrodotoxin resistance in pufferfishes. Curr. Biol. 15, 2069-2072]. The latter function remained to be elucidated. Thus, we further explored the function of these two residues, electrophysiologically, by evaluating the K-d (dissociation constants) values of TTX for F385N, F385A, F385Q, G1718T, and F385N/GI718T mutants of rNa(v)1.2a, transiently expressed in HEK-293 cells F385N caused 3000-fold increase of the K-d, while G1718T and F385N/GI718T caused 2- and 3-fold increases compared with those of WT and F385N, respectively, suggesting that G1718T further enhanced TTX resistivity caused by F385N. The K-d for F385A and F385Q were 2- and 11-fold larger than that of F385N, respectively, suggesting that the longer side chain in the non-aromatic amino acid residue causes the larger decrease of TTX sensitivity. Despite drastic changes in the K-d, the mutations at F385 caused only small changes in the k(off) from that of WT, suggesting that the Kd for TTX receptors are mainly determined by the k(on). (c) 2007 Elsevier Ltd. All rights reserved.