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Charge conversion enables quantification of the proximity between a normally‐neutral μ‐conotoxin (GIIIA) site and the Na + channel pore
Author(s) -
Li Ronald A,
Sato Kazuki,
Kodama Kyoko,
Kohno Toshiyuki,
Xue Tian,
Tomaselli Gordon F,
Marbán Eduardo
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)03316-6
Subject(s) - chemistry , stereochemistry , toxin , alanine , mutant , crystallography , allosteric regulation , sodium channel , residue (chemistry) , binding site , biophysics , amino acid , receptor , sodium , biochemistry , biology , organic chemistry , gene
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) (ΔΔ G =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.