Charge conversion enables quantification of the proximity between a normally‐neutral μ‐conotoxin (GIIIA) site and the Na+ channel pore

FEBS Letters

Li, Ronald A¹  Tomaselli, Gordon F¹  Kodama, Kyoko²  Sato, Kazuki²  Marbán, Eduardo¹  Xue, Tian¹  Kohno, Toshiyuki³ 

[1]Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue/Ross 844, Baltimore, MD 21205, USA

[2]Fukuoka Womens University, Fukuoka 813-8529, Japan

[3]Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194-8511, Japan

关键词: Sodium channel;    μ-Conotoxin;    Protein engineering;    Mutagenesis;    Mutant cycle analysis;   

DOI  :  10.1016/S0014-5793(01)03316-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

μ-Conotoxin (μ-CTX) inhibits Na⁺ flux by obstructing the Na⁺ channel pore. Previous studies of μ-CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C-terminal neutral alanine (A22) of μ-CTX and the Na⁺ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) μ-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced μ-CTX affinity for WT μ1 Na⁺ channels (90-fold↓), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT μ-CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (ΔΔG=2.2±0.1 vs. <1 kcal/mol for others). We conclude that μ-CTX residue 22 closely associates with the DII pore in the toxin-bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na⁺ channel pore.

【 授权许可】

Unknown