M. Chahine et al., CHARACTERIZING THE MU-CONOTOXIN BINDING-SITE ON VOLTAGE-SENSITIVE SODIUM-CHANNELS WITH TOXIN ANALOGS AND CHANNEL MUTATIONS, Receptors & channels, 3(3), 1995, pp. 161-174
The three-dimensional organization of the domains of the rat skeletal
muscle sodium channel subtype 1 (rSkM1) and the toxin-channel interact
ion surface have been explored by a complementary mutagenesis approach
. This method involves probing mutant channels with analogs of the pep
tide toxin, mu-conotoxin (mu-CTX), for which the tertiary structure ha
s been determined. mu-CTX has an overall net charge of +5. The blockin
g of Na+ currents of rSkM1 expressed in Xenopus oocytes by mu-CTX anal
ogs 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 po
tencies of up to 228-fold compared with an IC50 = 51.4+/-2.2 nM for na
tive 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 critic
al. 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 similar to 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 al more th
an a dozen mostly anionic sites in putative extracellular residues of
rSkM1. The toxin binding results indicate: firstly, many channel mutat
ions at anionic sidechains on the putative extracellular surface of mu
-CTX-sensitive channels, thought to be possible sites of interaction w
ith toxin, have been shown to have no effect on toxin binding. Secondl
y, 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 i
n IC50 value. The ratio of toxin blocking potencies was not significan
tly different when wild-type and the mutant (Tyr401Cys) rSkM1 channels
were studied with two toxin analogs, Arg19Gln and Arg13Gln, in contra
st to all other toxin derivatives examined. Since Tyr401 is known to b
e in the channel pore, these results suggest that either or both of th
e Arg residues at positions 13 and 19 of mu-CTX interact(s) with resid
ue Tyr401 of rSkM1 and, therefore, indicate that mu-CTX extends into t
he pore region of the channel.