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 + 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.
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