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

Ronald A. Li, Kazuki Sato, Kyoko Kodama, Toshiyuki Kohno, Tian Xue, Gordon F. Tomaselli, Eduardo Marbán

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)159-164
Number of pages6
JournalFEBS Letters
Volume511
Issue number1-3
DOIs
StatePublished - Jan 30 2002
Externally publishedYes

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Conotoxins
Glutamic Acid
Alanine
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Fluxes

Keywords

  • μ-Conotoxin
  • Mutagenesis
  • Mutant cycle analysis
  • Protein engineering
  • Sodium channel

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Charge conversion enables quantification of the proximity between a normally-neutral μ-conotoxin (GIIIA) site and the Na+ channel pore. / Li, Ronald A.; Sato, Kazuki; Kodama, Kyoko; Kohno, Toshiyuki; Xue, Tian; Tomaselli, Gordon F.; Marbán, Eduardo.

In: FEBS Letters, Vol. 511, No. 1-3, 30.01.2002, p. 159-164.

Research output: Contribution to journalArticle

Li, Ronald A. ; Sato, Kazuki ; Kodama, Kyoko ; Kohno, Toshiyuki ; Xue, Tian ; Tomaselli, Gordon F. ; Marbán, Eduardo. / Charge conversion enables quantification of the proximity between a normally-neutral μ-conotoxin (GIIIA) site and the Na+ channel pore. In: FEBS Letters. 2002 ; Vol. 511, No. 1-3. pp. 159-164.
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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. <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.

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