μ-ConotoxinGIIIA,一种从地理圆锥体分离的肽毒素,优先阻断骨骼肌钠通道NaV1.4。GIIIA紧密折叠成通过三个二硫键稳定的金字塔结构。为了评估GIIIA的单个二硫键对NaV1.4阻断的贡献,制备并表征了7种二硫键缺乏的类似物,每个人都有一个,两个,或三对二硫键结合的Cys残基替换为Ala。通过全细胞膜片钳对在HEK293细胞中异源表达的rNaV1.4测定类似物对NaV1.4的抑制效力。GIIIA的相应IC50值为0.069±0.005μM,GIIIA-1为2.1±0.3μM,GIIIA-2为3.3±0.2μM,GIIIA-3为15.8±0.8μM(-1,-2和-3分别表示二硫键Cys3-Cys15,Cys4-Cys20和Cys10-Cys21的去除)。其他类似物的活性不足以测量IC50。我们的结果表明,GIIIA的所有三个二硫键都是产生有效抑制NaV1.4所必需的,并且任何一个的去除都显着降低了其钠通道结合亲和力。Cys10-Cys21对于NaV1.4效力是最重要的。
μ-Conotoxin GIIIA, a peptide toxin isolated from Conus 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 IC50 values 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 IC50 measurement. 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.