The three-dimensional organization of the domains of the rat skeletal muscle sodium channel subtype 1 (rSkM1) and the toxin-channel interaction surface have been explored by a complementary mutagenesis approach. This method involves probing mutant channels with analogs of the peptide toxin, mu-conotoxin (mu-CTX), for which the tertiary structure has been determined. mu-CTX has an overall net charge of +5. The blocking of Na+ currents of rSkM1 expressed in Xenopus oocytes by mu-CTX analogs in which negative charge had been removed by Asn substitution for Asp or positive charge had been decreased by Gln substitution for Arg or Lys was studied; the mu-CTX analogs exhibited decreased blocking potencies of up to 228-fold compared with an IC50 = 51.4 +/- 2.2 nM for native mu-CTX on wild-type rSkM1. Mutations at Arg 13 of mu-CTX were the most critical in decreasing potency and at Lys9 were the least critical. Charge alone, however, was not the essential factor in some toxin substitutions: the IC50 value for Asp12Asn showed little change while that for Asp12Glu was increased approximately 100-fold due to a change in conformation (revealed by NMR measurements of the toxin in solution). Focusing on the sites in the channel which might be involved in toxin binding, mutations were introduced involving substitutions at more than a dozen mostly anionic sites in putative extracellular residues of rSkM1. The toxin binding results indicate: firstly, many channel mutations at anionic sidechains on the putative extracellular surface of mu-CTX-sensitive channels, thought to be possible sites of interaction with toxin, have been shown to have no effect on toxin binding. Secondly, one channel mutation, rSkM1/Tyr401Cys, (in the loop between S5 and S6 of Domain 1), affected mu-CTX potency causing a 3.7-fold increase in IC50 value. The ratio of toxin blocking potencies was not significantly different when wild-type and the mutant (Tyr401Cys) rSkM1 channels were studied with two toxin analogs, Arg19Gln and Arg13Gln, in contrast to all other toxin derivatives examined. Since Tyr401 is known to be in the channel pore, these results suggest that either or both of the Arg residues at positions 13 and 19 of mu-CTX interact(s) with residue Tyr401 of rSkM1 and, therefore, indicate that mu-CTX extends into the pore region of the channel.