A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule.
We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel.
Dudley
S C
SC
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
Todt
H
H
Lipkind
G
G
Fozzard
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D020916:Conotoxins; D004351:Drug Resistance; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010456:Peptides, Cyclic / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017354:Point Mutation; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D013779:Tetrodotoxin / Q000378:metabolism
D020916:Conotoxins; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; D015222:Sodium Channels; C046101:conotoxin GIII; D012530:Saxitoxin; D013779:Tetrodotoxin