Potassium Channels: from Scorpion Venoms to High-Resolution Structure
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Toxicon 39 (2001) 739±748 Review www.elsevier.com/locate/toxicon Potassium channels: from scorpion venoms to high-resolution structure M.L. Garciaa,*, Ying-Duo Gaob, O.B. McManusa, G.J. Kaczorowskia aDepartment of Membrane Biochemistry and Biophysics, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA bDepartment of Molecular Systems, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA Received 8 June 2000; accepted 5 July 2000 1. Introduction the efforts of the many research laboratories that have focused on the study of K1 channels: (1) the extensive clon- In 1998, the ®eld of ion channel research entered a new ing and functional expression of these proteins; and (2) the era when the ®rst, high-resolution crystal structure of one of existence of a large number of high af®nity peptidyl inhibi- these proteins was solved (Doyle et al., 1998). For the ®rst tors of these proteins, isolated from different scorpion and time, it was possible to understand, at a molecular level, the spider venoms (Tytgat et al., 1999). In fact, the use of pep- mechanisms that control ion selectivity and conduction in tidyl inhibitors derived from scorpion venoms provided the potassium channels. The protein whose structure had been ®rst indirect information concerning K1 channel structure. determined, the KcsA K1 channel, is a two transmembrane For instance, both identi®cation of the pore region of the spanning domain, potassium selective channel from Strepto- channel (MacKinnon and Miller, 1989b), and determination myces lividans that gates in response to H1 when reconsti- of the tetrameric composition of K1 channels (MacKinnon, tuted in arti®cial lipid bilayers (Cuello et al., 1998; 1991) were made possible with the use of scorpion toxins. In Heginbotham et al., 1999). The physiologic role of the addition, these peptides have been invaluable tools for puri- KcsA channel in Streptomyces is not well understood. The fying channels from native tissues, as well as determining amino acid sequence of the KcsA channel is closely related their subunit composition (Garcia-Calvo et al., 1994), and to that of some eukaryotic, six-transmembrane spanning also for understanding the physiological role of speci®c domain, potassium channels (Doyle et al., 1998). In parti- channel proteins (Garcia et al., 1997). Clearly, scorpion cular, the sequence in the pore region, containing the K1 toxins have played important, crucial roles in determining channel signature sequence, is almost identical to that found potassium channel structure and function. In this review, in vertebrate voltage-gated K1 channels. An exciting aspect we will discuss how work with scorpion venom peptides of the KcsA channel study was the ®nding that a number of have progressed from their initial discovery as K1 channel structural features predicted from functional studies with blockers, to their prominent role in interpreting the structure eukaryotic six-transmembrane domain voltage-gated K1 of the KcsA channel, and to the role that they may play in channels were present in the determined structure of the the future. Other aspects of work with these inhibitors, such prokaryotic channel. This provided compelling evidence as those concerning their role in developing the molecular that the overall architecture of K1 channels may be pharmacology of potassium channels have previously been conserved through evolution, and that the structure of the discussed extensively (Garcia et al., 1997; Garcia and KcsA channel can be used as a paradigm to probe the Kaczorowski, 1998), and, therefore, will not be reviewed properties of other K1 channels. here. The interpretation of structural features of the KcsA channel was greatly facilitated by a large amount of func- tional data accumulated in previous years with several 2. Potassium channel blocking peptides eukaryotic K1 channels. Two major factors have facilitated All potassium channel blocking peptides that have been puri®ed from scorpion venoms contain 30±40 amino acids * Corresponding author. Tel.: 11-732-594-7564; fax: 11-732- with three or four disul®de bridges. Based on primary 594-3925. sequence homology, 12 sub-families containing 49 different E-mail address: [email protected] (M.L. Garcia). peptides, termed a-KTX1±12, have been described (Tytgat 0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0041-0101(00)00214-2 740 M.L. Garcia et al. / Toxicon 39 (2001) 739±748 Fig. 1. Comparison of the amino acid sequences of charybdotoxin (ChTX; a-KTX1.1), L. quinquestriatus toxin 2 (Lq2; a-KTX1.2), iberiotoxin (IbTX, a-KTX1.3), noxiustoxin (NxTX; a-KTX2.1), margatoxin (MgTX; a-KTX2.2), hongotoxin1 (HgTX1; a-KTX2.5), kaliotoxin (KTX; a-KTX3.1), and agitoxin2 (AgTX2; a-KTX3.2) (Tytgat et al., 1999). The sequences have been aligned with respect to the six cysteine residues which are highlighted. The position of the disul®de bonds is indicated. Peptides are grouped in subfamilies according to their sequence homologies. et al., 1999). Out of these 49 peptides, only a restricted of channel gating (MacKinnon and Miller, 1988, 1989a; number have been studied in detail with respect to their Miller, 1988; Giangiacomo et al., 1992). Binding of the 1 interaction with K channels. Noxiustoxin, a-KTX2.1, puri- peptides occurs through a reversible, bimolecular reaction ®ed from venom of the scorpion Centruroides noxius in which is governed by electrostatic interactions between 1982, was the ®rst identi®ed K1 channel blocking peptide negatively charged residues in the channel and positively (Possani et al., 1982). Charybdotoxin (ChTX; a-KTX1.1), charged residues in the peptide. Consequently, the associa- identi®ed in 1985 as a component of Leiurus quinquestria- tion rate constant for binding of the peptide to the channel tus var. hebraeus venom, became the standard K1 channel increases as the ionic strength of the external media is inhibitory peptide (Miller et al., 1985; Garcia et al., 1995). diminished. Evidence supporting the idea that such peptides Subsequently, iberiotoxin (IbTX; aKTX1.3) (Galvez et al., function as pore blockers comes from two types of experi- 1990), margatoxin (MgTX; a-KTX2.2) (Garcia-Calvo et al., ments. The ®rst type of evidence comes from the interaction 1993), kaliotoxin (KTX; a-KTX3.1) (Romi et al., 1993), and between peptide toxins and TEA, which blocks some potas- agitoxin-2 (AgTX2; a-KTX3.2) (Garcia et al., 1994) (Fig. 1) sium channels by binding to a site in the pore close to the were found to interact with higher af®nity and speci®city at external entrance (Villarroel et al., 1988). TEA inhibits the some types of K1 channels, and these peptides became rate of association of ChTX and IbTX with some potassium prototype inhibitors of their respective target channels. channels in proportion with channel occupancy by TEA, The three-dimensional structure of a number of these but has no effect on toxin dissociation rate (Miller, 1988; peptides has been solved in solution by NMR techniques Giangiacomo et al., 1992). This ®nding suggests that the (Bontems et al., 1991, 1992; Johnson and Sugg, 1992; binding sites for these ligands overlap. In the second type, Fernandez et al., 1994; Johnson et al., 1994; Dauplais et the rate of toxin dissociation from the channel was deter- al., 1995; Krezel et al., 1995). All of these peptides adopt mined in the presence of different concentrations of mono- a backbone conformation consisting of three anti-parallel valent cations present at the inner face of the channel. The beta strands linked to an a-helical region by disul®de off rates of the toxins increased as the concentrations of bonds (Fig. 2). This folding pattern causes the molecule to permeant monovalent cations on the internal side was have a very compact structure where all residues, except for increased (MacKinnon and Miller, 1988; Giangiacomo et the cysteines, are exposed to solvent. The best characterized al., 1992; Goldstein and Miller, 1993). These data suggest peptides (ChTX, IbTX and AgTX2) interact with members that permeant ions occupy a site along the conduction path- 1 of the Kv1 family of voltage-gated K channels and/or way that is in close physical proximity to the receptor site calcium-activated potassium channels (Garcia et al., 1997). for the peptide. Occupancy of that site by permeant ions Mechanistic studies carried out in different laboratories destabilizes inhibitor binding, presumably by electrostatic clearly identify these peptides as pore blockers. A good repulsion with a positive charge on the toxin molecules. body of evidence indicates that all the peptides bind in the The positive charge on ChTX that mediates this effect has outer vestibule of the channel and block ion conduction by been localized to the side chain of Lys27 (Park and Miller, physically occluding the pore, without affecting the kinetics 1992a,b; Goldstein and Miller, 1993). M.L. Garcia et al. / Toxicon 39 (2001) 739±748 741 Fig. 2. Backbone structure of AgTX2. The solution structure used for the display was from (Krezel et al., 1995). The side chains of Arg24, Lys27, and Asn30 are shown. These residues when individually mutated to Ala cause a large destabilization on the peptide's interaction with the Shaker K1 channel (Ranganathan et al., 1996). Colors represent residues with the following properties: white, hydrophobic; green, polar; red, negatively charged; blue, positively charged; gray, aromatic; yellow, cysteine. Understanding the pore-blocking mechanism of ChTX interaction with potassium channels. When the S5±S6 linker and related peptides provided a powerful tool to identify was transferred from the highly toxin-sensitive Kv1.3 channel channel residues that form the pore and the external surface to the toxin-insensitive Kv2.1 potassium channel, the resulting surrounding the pore. The ®rst indications that the Shaker chimeric channel acquired sensitivity to AgTX2 (Gross et al., potassium channel pore region may be formed by residues 1994).