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

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, Na(v)1.4) Na(+) channels. mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by similar to20-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 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.