Tarantula toxins interacting with voltage sensors in potassium channels

Abstract

Voltage-activated ion channels open and close in response to changes in membrane voltage, a process that is crucial for electrical signaling in the nervous system. The venom from many poisonous creatures contains a diverse array of small protein toxins that bind to voltage-activated channels and modify the gating mechanism. Hanatoxin and a growing number of related tarantula toxins have been shown to inhibit activation of voltage-activated potassium (K_v) channels by interacting with their voltage-sensing domains. This review summarizes our current understanding of the mechanism by which these toxins alter gating, the location of the toxin receptor within K_v channels and the disposition of this receptor with respect to the lipid membrane. The conservation of tarantula toxin receptors among voltage-activated ion channels will also be discussed.

Introduction

Voltage-activated ion channels open and close in response to changes in membrane voltage, a process that underlies their fundamental roles in the generation and propagation of electrical signals within the nervous system. Not surprisingly, these crucial ion channels are popular targets for the wide array of protein toxins found in the venom of poisonous creatures. For example, venom from arachnids (spiders and scorpions), anthozoans (sea anemone), mollusks (cone snails) and reptiles (snakes) contain toxins that bind to voltage-activated potassium (K_v), sodium (Na_v) and calcium (Ca_v) channels (Possani et al., 2000; Rash and Hodgson, 2002; Srinivasan et al., 2002; Terlau and Olivera, 2004). The most widely appreciated biological function of toxins targeting voltage-activated channels is to paralyze prey, either by inhibiting (Na_v and Ca_v inhibitors) or excessively stimulating (K_v inhibitors and Na_v activators) transmission at synapses within the nervous system.

The toxins that interact with voltage-activated ion channels can be thought of as working through two distinct targeting mechanisms. In the first mechanism the toxin binds to the outer vestibule of the ion conduction pore and inhibits the flow of ions. The best studied examples of this mechanism are the scorpion toxins charybodotoxin and agitoxin (Miller, 1995), two particularly interesting K_v channel inhibitors that were used to find the pore forming region of potassium channels (MacKinnon and Miller, 1989). In the second type of mechanism the toxin binds to a region of the channel that changes conformation during gating and influences the gating mechanism by altering the relative stability of closed, open or inactivated states. The α- and β-scorpion Na_v channel toxins from Centruroides sculpturatus scorpion venom were the earliest studied protein toxins that modify gating of voltage-activated ion channels (Koppenhofer and Schmidt, 1968a, Koppenhofer and Schmidt, 1968b; Cahalan, 1975; Wang and Strichartz, 1983). This review primarily focuses on the more recently discovered family of toxins that modify gating of K_v channels, with occasional reference to related studies on toxins that interact with Ca_v and Na_v channels.

The X-ray structure of the mammalian K_v1.2 channel (Long et al., 2005) nicely illustrates the two primary regions of the channel that are targeted by toxins (Fig. 1). As the structure shows, K_v channels are tetramers, a fact that was first established from experiments with the pore blocking scorpion toxin, agitoxin (MacKinnon, 1991). Each K_v channel subunit contains 6 transmembrane segments, termed S1–S6, arranged into two types of domains; a single pore domain formed by the S5–S6 regions from the four subunits (yellow helices), and four surrounding voltage-sensing domains, each one formed by the S1–S4 helices from a single subunit. The pore domain houses the K⁺ selective ion conduction pathway (demarcated by the red potassium ions in Fig. 1B,C), and the receptor for pore blocking toxins that bind to the extracellular vestibule near the selectivity filter. In the case of charybdotoxin, a Lys residue on the active surface of the toxin projects into the pore of the channel and interacts with potassium ions bound within the selectivity filter (Anderson et al., 1988; MacKinnon and Miller, 1988; Park and Miller, 1992). Many other types of K⁺ channels only contain the equivalent of the pore domain of K_v channels (Jan and Jan, 1997), and pore blocking scorpion and bee toxins similarly bind to the extracellular entrance of the pore in these simpler channels (Lu and MacKinnon, 1997; Jin and Lu, 1998), highlighting the conserved nature of potassium channel pores. The pore domain also contains the S6 activation gate, an intracellular region of the ion conduction pore that prevents the flow of ions in the closed state (Fig. 1B) (Yellen, 2002). The S6 gate region of the protein does not appear to be directly targeted by protein toxins, presumably because poisonous creatures deposit their venom into the extracellular solution and the S6 gate is located on the opposite side of the membrane.

The voltage-sensing domain is a second region of the channel that is widely targeted by toxins, in this case by the class of toxins that bind to and inhibit the channel by stabilizing a particular state in the gating pathway. The process of voltage-dependent gating in K_v channels is thought to involve several relatively independent motions of the four voltage-sensing domains between resting (R) and activated (A) states, followed by a concerted opening transition where the S6 gate moves from a closed (C) to an open (O) state (Scheme 1; (Ledwell and Aldrich, 1999)). There is growing evidence to suggest that the S4 segment within the voltage-sensing domains also moves during the concerted opening transition (Smith-Maxwell et al., 1998a, Smith-Maxwell et al., 1998b; Ledwell and Aldrich, 1999; del Camino et al., 2005; Pathak et al., 2005), and therefore a toxin that binds to the voltage-sensing domains could in principle inhibit channel activation by stabilizing any of the states prior to the open state.

Hanatoxin is the founding member of a family of toxins that bind to the voltage sensing domains in K_v channels and inhibit opening of these channels. This 35 residue protein toxin was originally isolated from the venom of the Chilean rose tarantula (Grammostola spatulata) during a search for new inhibitors of the K_v2.1 channel (Swartz and MacKinnon, 1995). The selectivity of hanatoxin is far from absolute, as it also inhibits K_v4.2 channels with similar affinity compared to K_v2.1 (see also Section 5.1). Although hanatoxin is one of the most extensively studied toxins targeting the voltage sensing domains in K_v channels (see below), the list of newer toxins in this class is rapidly growing (Fig. 2). The closely related SGTx1 from Scodra griseipes (Marvin et al., 1999; Lee et al., 2004) also inhibits K_v2.1 and systematic mutagenesis on this toxin has identified the active face of the molecule (Wang et al., 2004). The recently discovered guangxitoxin (GxTx1E) from Plesiophrictus guangxiensis has particularly high affinity for K_v2.1 (Herrington et al., 2006), which should open up promising opportunities for better understanding the complex between this family of toxins and voltage-sensing domains (see Section 2.3). Stromatoxin (ScTx1) from Stromatopelma calceata and heteroscodratoxins (HmTx1,2) from Heteroscodra maculate target both K_v2 and K_v4 channels (Escoubas et al., 2002), whereas the heteropodatoxins (HpTx1-3) from Heteropoda venatoria (Sanguinetti et al., 1997; Zarayskiy et al., 2005), phrixotoxins (PaTx1,2) from Phrixotrichus auratus (Diochot et al., 1999) and TLTx1-3 from Theraphosa leblondi venom (Ebbinghaus et al., 2004) seem to primarily target K_v4 channels. One of the more recent additions to this family of toxins is VsTx1, a toxin that was isolated from Grammostola spatulata venom when screening for activity against the K_vAP channel (Ruta et al., 2003; Ruta and MacKinnon, 2004), an archebacterial K_v channel for which several X-ray crystal structures are available (Jiang et al., 2003a; Lee et al., 2005). The amino acid sequences of the K_v channel toxins discussed thus far show varying similarity, ranging from 82% to 30%, and all have six cysteine residues that form 3 disulfide bonds at the core of the molecules (Fig. 2; see also Section 3). It is rather interesting that with the exception of HpTx1-3, all of these K_v channel gating modifier toxins were isolated from tarantula venom. Although it is a mystery why these particular creatures are so fond of producing gating modifier toxins (and why they appear uninterested in making pore blocking protein toxins), it is clear that their venom is a particularly rich and relatively untapped repository of these toxins.

It has recently become evident that sea anemone also produce gating modifier toxins that interact with K_v channels. BDS I and II from Anemonia sulcata inhibit K_v3 channels (Diochot et al., 1998) and the related APETx1 from Anthopleura elegantissima venom inhibit HERG K_v channels (Diochot et al., 2003; Restano-Cassulini et al., 2006). The sequences of these anemone toxins are quite different than other K_v channel toxins discussed here, but a growing body of work suggests that they probably inhibit through a related mechanism (Diochot et al., 2003; Yeung et al., 2005).