ACTION OF DERIVATIVES OF MU-CONOTOXIN-GIIIA ON SODIUM-CHANNELS - SINGLE AMINO-ACID SUBSTITUTIONS IN THE TOXIN SEPARATELY AFFECT ASSOCIATION AND DISSOCIATION RATES
ACTION OF DERIVATIVES OF MU-CONOTOXIN-GIIIA ON SODIUM-CHANNELS - SINGLE AMINO-ACID SUBSTITUTIONS IN THE TOXIN SEPARATELY AFFECT ASSOCIATION AND DISSOCIATION RATES
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DOI：
10.1021/bi00150a016
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发表时间：
1992-09-08
期刊：
影响因子：
2.9
通讯作者：
FRENCH, RJ
中科院分区：
文献类型：
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作者：
BECKER, S;PRUSAKSOCHACZEWSKI, E;FRENCH, RJ
本文研究了22个氨基酸残基的魔芋毒素GIIIA(MU-CTX)的12个衍生物对钠通道的结合和阻断作用，其中GLN取代Arg或Lys，Lys取代Gln-18，Asn取代Asp，Pro取代HO-Pro。按照Becker等人的描述合成了衍生物。[(1989)欧元。J.生物化学。185，79]。通过置换鳗鱼电塑膜(100 mM氯化胆碱，10 mM HEPES-NaOH，pH 7.4)上标记的岩藻毒素来测定结合能力。通过稳态、单通道记录，将大鼠骨骼肌钠通道结合到平面中性磷脂/正庚烷双层膜(200 mM NaC l，10 mM HEPES-NaOH，pH 7.0)中，评估阻断动力学。阻塞事件通常表现为几秒到几分钟的时间段，在这段时间内通过单个通道的电流完全消除。一个值得注意的例外是替代Arg-13-Gln，对于该替代Arg-13-Gln，“阻断”事件显示的可测量电导约为开放状态的20%-40%。Arg-13的替代将与电浆膜的结合减少到无法检测的水平，并使骨骼肌通道的表观解离常数增加了>；80倍于天然多肽。其他替换导致亲和力较小的下降。毒素衍生物效力的降低既是由于通道解离速率的增加，也是由于结合率的降低。我们的数据支持佐藤等人的建议。[(1991)J.Biol.化学。265,16989]Arg-13与通道上的结合位点紧密结合。此外，我们的结果表明，某些残基几乎只影响毒素与其结合部位的接近和对接，其他残基似乎只有在结合发生后才对结合的强度重要，而另一些残基则同时影响两者。
We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185,79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by >80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.