A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B.
The polypeptide neurotoxin anthopleurin B (ApB) isolated from the venom of the sea anemone Anthopleura xanthogrammica is one of a family of toxins that bind to the extracellular face of voltage-dependent sodium channels and retard channel inactivation. Because most regions of the sodium channel known to contribute to inactivation are located intracellularly or within the membrane bilayer, identification of the toxin/channel binding site is of obvious interest. Recently, mutation of a glutamic acid residue on the extracellular face of the fourth domain of the rat neuronal sodium channel (rBr2a) was shown to disrupt toxin/channel binding (Rogers, J. C., Qu, Y. S., Tanada, T. N., Scheuer, T., and Catterall, W. A. (1996) J. Biol. Chem. 271, 15950-15962). A negative charge at this position is highly conserved between mammalian sodium channel isoforms. We have constructed mutations of the corresponding residue (Asp-1612) in the rat cardiac channel isoform (rH1) and shown that the lowered affinity occurs primarily through an increase in the toxin/channel dissociation rate koff. Further, we have used thermodynamic mutant cycle analysis to demonstrate a specific interaction between this anionic amino acid and Lys-37 of ApB (DeltaDeltaG = 1.5 kcal/mol), a residue that is conserved among many sea anemone toxins. Reversal of the charge at Asp-1612, as in the mutant D1612R, also affects channel inactivation independent of toxin (-14 mV shift in channel availability). Binding of the toxin to Asp-1612 may therefore contribute both to toxin/channel affinity and to transduction of the effects of the toxin on channel kinetics.
Benzinger
G R
GR
Department of Committee on Neurobiology, The University of Chicago, Chicago, Illinois 60637, USA.
Kyle
J W
JW
Blumenthal
K M
KM
Hanck
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D015222:Sodium Channels; C026132:anthopleurin B