期刊
FEBS LETTERS
卷 511, 期 1-3, 页码 159-164出版社
WILEY
DOI: 10.1016/S0014-5793(01)03316-6
关键词
sodium channel; mu-conotoxin; protein engineering; mutagenesis; mutant cycle analysis
资金
- NHLBI NIH HHS [R01 HL-52768, R01 HL-50411] Funding Source: Medline
mu-Conotoxin (mu-CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of mu-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 mu-CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) mu-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced mu-CTX affinity for WT mu1 Na+ channels (90-fold down arrow), 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 mu-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) (DeltaDeltaG = 2.2+/-0.1 vs. < 1 kcal/mol for others). We conclude that mu-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. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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