title: 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.
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.
authors.affiliation: Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. ronaldli@jhmi.edu
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
authors.forename: Kwokyin
authors.initials: K
authors.lastname: Hui
authors.forename: Robert J
authors.initials: RJ
authors.lastname: French
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
authors.forename: Charles A
authors.initials: CA
authors.lastname: Henrikson
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
authors.forename: Eduardo
authors.initials: E
authors.lastname: Marbán
mesh_terms: D000818:Animals; D001120:Arginine / Q000235:genetics; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020916:Conotoxins / Q000494:pharmacology*; D018698:Glutamic Acid / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects*; D008239:Lysine / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000493:pharmacokinetics*; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002121:Calcium Channel Blockers; D020916:Conotoxins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D018698:Glutamic Acid; D001120:Arginine; D012964:Sodium; D008239:Lysine