Butterworth J F, Moran J R, Whitesides G M, Strichartz G R
J Med Chem. 1987 Aug;30(8):1295-302. doi: 10.1021/jm00391a006.
To measure the depth of the local anesthetic binding site within the neuronal membrane, biotin-containing polyethylene glycols having zero, three, and six ethylene glycol subunits were added to the p-amino termini of tetracaine and procaine, thereby interposing a pharmacologically inert "spacer" molecule between the local anesthetic and the biotin moiety. These biotinyl-local anesthetic derivatives produced "tonic" inhibition of the compound action potential of split, desheathed frog sciatic nerves in a concentration-dependent, reversible manner. However, no inhibition of the action potential occurred when sufficient avidin, a 66,000-MW protein that binds four biotins, was present to bind and anchor the biotin-containing end of each derivative outside the plasma membrane. Increasing the "leashed" anesthetic derivative's concentration to 4 times that which reduced impulse height by 50% in the absence of avidin still produced no detectable block when equimolar avidin was present. Apparently, the "spacer" in the derivative compound was too short to permit the avidin-complexed anesthetic to reach its site of action on the sodium channel. In a similar fashion, the local anesthetic derivatives produced "use-dependent" block when drug-treated nerves were stimulated at 40 Hz in the absence of equimolar avidin, but failed to produce "use-dependent" block when equimolar avidin was present. In common with others, we assume that tertiary amine local anesthetics may reach their binding site via hydrophobic (transmembrane) pathways without necessarily entering the cytoplasm. Thus, since our longest local anesthetic derivative, that containing six ethylene glycol subunits, placed the local anesthetic group a maximum of 15-18 A from the surface of the avidin moiety, we conclude that the local anesthetic binding site for block of sodium channels of amphibian nerve must be greater than or equal to 15 A from the outer surface of the plasma membrane.
为了测量局部麻醉药在神经元膜内结合位点的深度，将含有零个、三个和六个乙二醇亚基的生物素化聚乙二醇添加到丁卡因和普鲁卡因的对氨基末端，从而在局部麻醉药和生物素部分之间插入一个药理惰性的“间隔”分子。这些生物素化局部麻醉药衍生物以浓度依赖性、可逆的方式对分离的、去鞘的青蛙坐骨神经的复合动作电位产生“强直”抑制。然而，当存在足够的抗生物素蛋白（一种结合四个生物素的66,000道尔顿蛋白质）以结合并将每个衍生物的含生物素末端锚定在质膜外时，动作电位没有受到抑制。当存在等摩尔抗生物素蛋白时，将“束缚”的麻醉药衍生物的浓度增加到在不存在抗生物素蛋白时使冲动高度降低50%的浓度的4倍，仍然没有产生可检测到的阻滞。显然，衍生物化合物中的“间隔物”太短，以至于抗生物素蛋白复合的麻醉药无法到达其在钠通道上的作用位点。以类似的方式，在不存在等摩尔抗生物素蛋白的情况下，当以40Hz刺激经药物处理的神经时，局部麻醉药衍生物产生“使用依赖性”阻滞，但当存在等摩尔抗生物素蛋白时则未能产生“使用依赖性”阻滞。与其他人一样，我们假设叔胺类局部麻醉药可能通过疏水（跨膜）途径到达其结合位点，而不一定进入细胞质。因此，由于我们最长的局部麻醉药衍生物（含有六个乙二醇亚基）使局部麻醉药基团距离抗生物素蛋白部分的表面最多15 - 18埃，我们得出结论，两栖神经钠通道阻滞的局部麻醉药结合位点必须距离质膜外表面大于或等于15埃。
