Dependence of mu-conotoxin block of sodium channels on ionic strength but not on the permeating [Na+]: implications for the distinctive mechanistic interactions between Na+ and K+ channel pore-blocking toxins and their molecular targets

J Biol Chem. 2003 Aug 15;278(33):30912-9. doi: 10.1074/jbc.M301039200. Epub 2003 May 21.

Abstract

Mu-conotoxins (mu-CTXs) are Na+ channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (mu), no such dependence has been reported for mu-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of mu and permeant ion concentration ([Na+]) on mu-CTX block of rat skeletal muscle (mu1, Nav1.4) Na+ channels. Mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by approximately 20-fold) when mu was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mm Na+); however, toxin block was unaltered (p > 0.05) when mu was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low mu was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k(on), as the primary determinant of the changes in affinity (k(on) increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external mu. Taken together, our findings indicate that mu-CTX block of Na+ channels depends critically on mu but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arginine / genetics
  • Calcium Channel Blockers / pharmacology
  • Conotoxins / pharmacology*
  • Glutamic Acid / genetics
  • Ion Channel Gating / drug effects*
  • Lysine / genetics
  • Membrane Potentials / drug effects
  • Muscle, Skeletal / metabolism
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Rats
  • Sodium / pharmacokinetics*
  • Sodium Channel Blockers / pharmacology*
  • Sodium Channels / chemistry
  • Sodium Channels / genetics
  • Sodium Channels / metabolism*

Substances

  • Calcium Channel Blockers
  • Conotoxins
  • Sodium Channel Blockers
  • Sodium Channels
  • Glutamic Acid
  • Arginine
  • Sodium
  • Lysine