TY - JOUR
T1 - Charge conversion enables quantification of the proximity between a normally-neutral μ-conotoxin (GIIIA) site and the Na+ channel pore
AU - Li, Ronald A.
AU - Sato, Kazuki
AU - Kodama, Kyoko
AU - Kohno, Toshiyuki
AU - Xue, Tian
AU - Tomaselli, Gordon F.
AU - Marbán, Eduardo
PY - 2002/1/30
Y1 - 2002/1/30
N2 - 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. + channel pore.
AB - 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. + channel pore.
KW - μ-Conotoxin
KW - Mutagenesis
KW - Mutant cycle analysis
KW - Protein engineering
KW - Sodium channel
UR - http://www.scopus.com/inward/record.url?scp=0037196391&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037196391&partnerID=8YFLogxK
U2 - 10.1016/S0014-5793(01)03316-6
DO - 10.1016/S0014-5793(01)03316-6
M3 - Article
C2 - 11821068
AN - SCOPUS:0037196391
VL - 511
SP - 159
EP - 164
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 1-3
ER -