A Specific Interaction between the Cardiac Sodium Channel and Site-3 Toxin Anthopleurin B

Abstract

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 k off . Further, we have used thermodynamic mutant cycle analysis to demonstrate a specific interaction between this anionic amino acid and Lys-37 of ApB (⌬⌬G ‫؍‬ 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.

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

“…ApB substantially enhances VGSC currents by selectively impairing fast inactivation (Benzinger et al, 1998). We found that ApB significantly enhanced the gating-pore currents generated in DIV (Fig.…”

Differential Phospholipid Binding by Site 3 and Site 4 Toxins

“…The pores of such channels have been mapped by analysis of their interactions with conotoxins (Na V ) and a variety of polypeptides from scorpion venoms, such as charybdotoxin and agitoxins (1)(2)(3)(4). More recently, regions of these channels involved in gating have been identified by using gating modifier toxins derived from scorpion (5), sea anemone (6), and spider (7)(8)(9) venoms. Most interestingly, gating modifier toxins appear to interact with the same channel region, designated the S3-S4 linker, irrespective of the type of channel being studied (10).…”

“…ApB is a 49-amino acid residue polypeptide toxin that functions to delay channel inactivation upon binding to site 3 of Na V s (13), where it competes with a number of scorpion ␣-toxins that have functionally identical effects. Site 3 has been mapped by mutant cycle analysis to the S3-S4 linker of Na V domain IV (5,6). ProTx-II (PT-II) is a recently discovered 30-amino acid residue polypeptide that conforms to the inhibitory cysteine knot motif and functions as a gating modulator to inhibit Na V activation (14).…”

Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5

“…These gating modifier toxins bind to sites 3 and 4, respectively. Site 3 has been localized to the extracellular S3/S4 linker of domain IV (9,10), whereas residues in domain II S3/S4 make a major contribution to site 4 (11,12).…”