title: The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of NaV1.4.
abstract: μ-Conotoxin GIIIA, a peptide toxin isolated fromConus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3-Cys15, Cys4-Cys20 and Cys10-Cys21, respectively). Other analogues were not active enough for IC50measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10-Cys21 is the most important for the NaV1.4 potency.
authors.affiliation: College of Science, National University of Defense Technology, Changsha 410073, Hunan, China. hanpeng1021@126.com.|Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. hanpeng1021@126.com.
authors.forename: Penggang
authors.initials: P
authors.lastname: Han
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. yiyongjun1949@163.com.
authors.forename: Kang
authors.initials: K
authors.lastname: Wang
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. daixiandong@139.com.
authors.forename: Xiandong
authors.initials: X
authors.lastname: Dai
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. cao_ying2002@hotmail.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. shangyiliu@sohu.com.
authors.forename: Shangyi
authors.initials: S
authors.lastname: Liu
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. jiangtide@sina.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. chongxu_fan@hotmail.com.
authors.forename: Chongxu
authors.initials: C
authors.lastname: Fan
authors.affiliation: College of Science, National University of Defense Technology, Changsha 410073, Hunan, China. wjwu67@126.com.
authors.forename: Wenjian
authors.initials: W
authors.lastname: Wu
authors.affiliation: College of Science, National University of Defense Technology, Changsha 410073, Hunan, China. chenjsh@cae.cn.|Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China. chenjsh@cae.cn.
authors.forename: Jisheng
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D002460:Cell Line; D020916:Conotoxins / Q000494:pharmacology*; D052078:Conus Snail / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D018482:Muscle, Skeletal / Q000187:drug effects; D011485:Protein Binding; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000378:metabolism*
chemical_list: D020916:Conotoxins; D004220:Disulfides; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C046101:conotoxin GIII
keywords: GIIIA; NaV1.4; disulfide bond; electrophysiology; μ-conotoxin