Choudhary Gaurav, Shang Lisa, Li Xiufeng, Dudley Samuel C
Department of Medicine, Emory University and Atlanta Veterans Administration Medical Center, Atlanta, Georgia 30322 USA.
Biophys J. 2002 Aug;83(2):912-9. doi: 10.1016/S0006-3495(02)75217-X.
Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional -OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na(+) channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC(50)s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains.
石房蛤毒素（STX）在由四个结构域的P环排列形成的外部前庭处选择性阻断电压门控钠通道。新石房蛤毒素在1,2,3 - 胍基的N1位置（N1 - OH）有一个额外的 - OH基团，它与Na(+)通道的结构域I和IV相互作用。确定毒素与通道的第二种相互作用将确定STX的位置。膝沟藻毒素2,3和膝沟藻毒素1,4分别是石房蛤毒素和新石房蛤毒素的C - 11硫酸化衍生物。我们利用这些变体通过在能量上定位外部前庭中的C - 11硫酸盐来限制STX的对接方向。通过一种系统的突变循环分析方法确定了C - 11硫酸盐与通道的四个结构域中每个结构域之间的相互作用，在该方法中，所有对1型毒素结合重要的已知羧基都被中和，从而实现对毒素硫酸盐的能量三角测量，并克服了突变循环的一些局限性。通过双电极电压钳测量注射了通道mRNA的非洲爪蟾卵母细胞的毒素IC(50)。基于耦合结果的三种独特分析类型将C - 11硫酸盐定位在结构域III和IV之间。结合我们之前的报告，这些数据确定了STX在外部前庭中的方向，并证实了通道结构域的顺时针排列。
