Differential Phospholipid Binding by Site 3 and Site 4 Toxins

Abstract

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na V 1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/ phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na V 1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K V AP channel than does the conformation of domain IV.Chemically diverse neurotoxins have historically been of great value in defining the overall architecture of voltage-dependent Na ϩ (Na V ) 1 and K ϩ (K V ) channels. 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 (1-4). 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 (7-9) 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).Gating modifier toxins can also be important probes for the accessibility of defined regions of a given channel. Very recently, the MacKinnon laboratory has employed a novel spider toxin, VSTX, to probe the accessibility of defined regions of the voltage sensor of the archaebacterial K V AP channel (9). The resulting data were interpreted in the context of a channel three-dimensional structure (11) in which the K V AP S3-S4 linker was located either near the cytoplasmic surface or buried within the bilayer, depending on whether the channel was in the resting or activated state (9). The observation that VSTX possessed phospholipid binding activity (12) provided a potential explanation for the ability of this toxin to modify channels via interaction with sequences that were never exposed at the extracellular surface.To understand the extent to which this model could ...

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

“…In contrast, dissociation remained a zero-order reaction as expected ( 250 nM ϭ 53.3 Ϯ 3.2 S, n ϭ 3; 500 nM ϭ 41.7 Ϯ 2.2 S, n ϭ 3). ProTx-II also shifts the voltage dependence of gating to more depolarized potentials, indicating that the toxin does not inhibit through a pore-blocking mechanism but, rather, interferes with the energetics of gating (15). Analysis of ProTx-II on steady-state activation and inactivation kinetics revealed that the midpoint of the activation curve shifted by 23 mV in the depolarizing direction, whereas inactivation remained unaffected by the toxin (Fig.…”

“…The cleaved product was then subcloned into an octahistidine version of the pMALc2x vector (New England Biolabs) between EcoRI and HindIII using standard molecular biology protocols. As described previously, two additional N-terminal amino acids derived from a StuI restriction site remain in the ProTx-II coding sequence (15). Site-directed mutagenesis was performed using the QuikChange method (Stratagene, La Jolla, CA) to create all recombinant toxin mutants described in this paper, and all constructs were verified by sequencing.…”

“…After dialysis, the proteins were oxidized by dropwise addition of GSSG to a final concentration of 0.5 mM and allowed to incubate for 72 h. Fusion proteins were then dialyzed against 50 mM NH 4 HCO 3 and cleaved overnight at room temperature with enterokinase (Novagen/EMD Biosciences). Toxins were purified via RP-HPLC as described (15). Molecular weights of purified toxins were confirmed by MALDI-TOF mass spectroscopy analysis on a Bruker Biflex IV spectrometer.…”

“…Wild-type and mutant toxins were expressed as fusion proteins containing maltose-binding protein upstream of the toxin in Escherichia coli BL21 (DE3) as described (15). After lysis in a French press, the supernatants obtained were purified on Ni 2ϩ -nitrilotriacetic acid resin and reduced with 10 mM dithiothreitol for 1 h at 37°C.…”

“…It is thought that the open probability of the channel increases because of the toxin locking the channel in its activated conformation after a depolarizing pre-pulse. ProTx-II is a 30-amino acid peptide toxin purified from the venom of the tarantula, Thrixopelma pruriens, that modifies activation of both Na v and Ca v , but not K v , channel isoforms by inhibiting peak current and shifting the voltage dependence of activation to more depo-larized potentials (14,15). ProTx-II conforms to the inhibitory cystine knot (ICK) 2 motif, a common structural fold among spider toxins targeting ion channels (see The KNOTTIN database online).…”