Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.
Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.
复制标题
通过从 Electrophorus electricus 的 electroplax 中分离出的电压调节钠通道重建神经毒素调节的离子传输。
DOI：
10.1073/pnas.81.4.1239
复制
发表时间：
1984
影响因子：
11.1
通讯作者：
Agnew,WS
中科院分区：
文献类型：
--
作者：
Rosenberg,RL;Tomiko,SA;Agnew,WS
本文报道了从Electrophorus electricus的电泳膜中纯化的电压调节钠通道的功能重建。通过用Bio-Beads SM-2去除去污剂，然后在添加脂质体的情况下进行冻融超声处理来实现复溶。该制剂显示3 H标记的河豚毒素（TTX）的热稳定结合（Kd = 33 nM）。22 Na+内流被生物碱神经毒素刺激2- 5倍，并被TTX阻断。藜芦定激活Na+内流的K1/2为18 μ M，这种激活被TTX精确地与特异性[3 H]TTX结合平行地阻断。Batrachotoxin刺激22 Na+通量比藜芦定更有效。没有发现肽海葵毒素II的影响。Na通道插入膜导致60-70%的TTX结合位点面向囊泡外部。因此，外部TTX部分抑制流量，而封锁是完全时，TTX也与囊泡内部平衡。脂溶性局麻药丁卡因和地布卡因完全抑制流量。QX-222是利多卡因的一种带电衍生物，仅阻断了一部分通道，显然是那些由内向外的通道。纯化样品主要由Mr 260，000 - 300，000糖肽组成，但含有可变量的较小肽。通过凝胶过滤柱配置文件确定了藜芦碱依赖性通量和肽组合物。受激通量仅与大肽共分布。
The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.