TY - JOUR
T1 - Dependence of μ-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
AU - Li, Ronald A.
AU - Hui, Kwokyin
AU - French, Robert J.
AU - Sato, Kazuki
AU - Henrikson, Charles A.
AU - Tomaselli, Gordon F.
AU - Marbán, Eduardo
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2003/8/15
Y1 - 2003/8/15
N2 - μ-Conotoxins (μ-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 (μ), no such dependence has been reported for μ-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of μ-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of μ and permeant ion concentration ([Na+]) on μ-CTX block of rat skeletal muscle (μ1, Nav1.4) Na + channels. μ-CTX sensitivity of wild-type and E758Q channels increased significantly (by ∼20-fold) when μ was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mM Na +); however, toxin block was unaltered (p > 0.05) when μ was maintained by replacement of [Na+] with N-methyl-D-glucamine (NMG+), suggesting that the enhanced sensitivity at low μ was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, kon, as the primary determinant of the changes in affinity (kon increased 40- and 333-fold for μ-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 μ-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external μ. Taken together, our findings indicate that μ-CTX block of Na+ channels depends critically on μ 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.
AB - μ-Conotoxins (μ-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 (μ), no such dependence has been reported for μ-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of μ-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of μ and permeant ion concentration ([Na+]) on μ-CTX block of rat skeletal muscle (μ1, Nav1.4) Na + channels. μ-CTX sensitivity of wild-type and E758Q channels increased significantly (by ∼20-fold) when μ was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mM Na +); however, toxin block was unaltered (p > 0.05) when μ was maintained by replacement of [Na+] with N-methyl-D-glucamine (NMG+), suggesting that the enhanced sensitivity at low μ was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, kon, as the primary determinant of the changes in affinity (kon increased 40- and 333-fold for μ-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 μ-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external μ. Taken together, our findings indicate that μ-CTX block of Na+ channels depends critically on μ 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.
UR - http://www.scopus.com/inward/record.url?scp=0042009353&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0042009353&partnerID=8YFLogxK
U2 - 10.1074/jbc.M301039200
DO - 10.1074/jbc.M301039200
M3 - Article
C2 - 12764145
AN - SCOPUS:0042009353
SN - 0021-9258
VL - 278
SP - 30912
EP - 30919
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 33
ER -