sign in sign up
<<
Home , Charybdotoxin, Kaliotoxin, Noxiustoxin

Neuron, Vol. 15, 5-10, July, 1995, Copyright 0 1995 by Cell Press The Family Review of K+ Channel-Blocking

Christopher Miller the , at far lower abundance than the major spe- Howard Hughes Medical Institute cies of toxic peptides, which are directed against Na+ chan- Graduate Department of Biochemistry nels. Electrophysiologically, the Na+ and K’channel Brandeis University cooperate with a determined depolarizing purpose: the Waltham, Massachusetts 02254 former by holding Na+ channels open, the latter by blocking the repolarizing K+ currents. The venoms massively dis- For many years, neuronal K’ channels were pharmacolog- rupt neuronal activity by producing repetitive firing fol- ical orphans, essential membrane proteins against which’ lowed by long-lived depolarization. Field ques- no natural toxins were known. While biochemically and tions about the biological role of specific peptides are physiologically useful high affinity ligands had been identi- difficult to approach systematically, since a typical scor- fied for Na’, Ca2+, and many ligand-gated channels, the pion contains, among its -50 distinct peptides, best blocker available for K+ channels was tetraethylam- several isoforms each of Na+ channel and K+ channel tox- monium. In 1982, this situation changed with the discovery ins Here is a speculation about the function of K+ channel by Possani and colleagues (Carbone et al., 1982; Possani toxins in a venom rich with Na+ channel toxins. The Na’ et al., 1982) of a in the venom of the channel toxins work by binding preferentially to, and thus Centruroides noxiusthat inhibited K+ currents in theclassi- stabilizing, the channel’s open state; the Na+ channel tox- cal electrophysiological preparation, the squid giant axon. ins are thus use dependent in action and require the neu- In the ensuing years, a large collection of homologous ron to be depolarized before binding can occur (Possani et peptides from was identified, and these were al., 1982; Catterall, 1977; Catterall, 1980). The K+ channel all found to block either of two major classes of K+ chan- toxins, however, are not so constrained and bind well to nels, voltage-gated (Kv-type) and high conductance Ca’+- both open and closed states of K+ channels (Anderson et activated (BK-type) K’ channels. These toxins inhibit in al., 1988; Park and Miller, 1992a; Goldstein and Miller, the 1 pM to 10 nM concentration range, and they act only 1993). These toxins, therefore, would act as initiators to on K+ channels. My purpose here is to summarize the elicit immediate depolarization, thus rendering the Na+ molecular characteristics of these toxins and to indicate channels susceptible to the abundant Na+ toxins in the the unique kinds of molecular information they have pro- venom. vided about the K+ channels that act as their receptors. I will not discuss any of the other classes of peptide toxins a-KTx Molecular Families against K’ channels now recognized, such as apamin All a-KTx peptides characterized so far share obvious se- (Hughes et al., 1982) leiurotoxin (Chicchi et al., 1988), or quence similarities (Table 1). All are 37-39 residues in (Parcej and Dolly, 1989; Hurst et al., 1991). length and have 6 easily aligned cysteine residues. The First, a word-boring but necessary-on the nomencla- last 3 cysteines participate in a conserved sequence, ture of these peptides, which have been named either which makes up much of the of the molecule: whimsically or according to the scorpion species in which the peptide is found. This is an unsatisfactory situation in Gze-[ *]~~~-Kz-CZ~-(M/I)~V-(NIG)~~-(X)~,-K~&& ( 2 )&&, view of the expanding list of homologs. Whimsical names provided an amusing mnemonic device when only where the charybdotoxin numbering convention is used, a handful of such toxins had been identified, but our limited [ *] represents a gap or a small residue, ( f ) is a charged imaginations cannot sustain this practice for long; species- residue, and (X) is variable. based names present a double difficulty, since each Closer examination of the sequences alone, combined venom is packed with multiple isoforms, and since very with a little subjective hairsplitting, shows that the toxins similar toxins are found in widely different species. Accord- fall into at least three subfamilies. Charybdotoxin-like tox- ingly, I suggest a formal name for this family of scorpion ins, subfamily 1 in Table 1, all have F at position 2 and venom peptides: the “a-K-toxins,” abbreviated “a-KTxmn,” W at position 14. Likewise, the -like peptides using a subfamily nomenclature that mirrors modern us- (subfamily 2) have a small-residue insertion between G26 age for the K+ channels themselves. Thus, according to and K27, while -like peptides (subfamily 3) dis- the molecular subfamilies shown in Table 1, charybdotoxin play a point deletion between the third and fourth cysteines will be called a-KTxl .l, noxiustoxin a-KTx2.1, 2 (probably at position 21). a-KTx3.2, etc. The functional specificities of the toxins support the use- fulness of these sequence-based distinctions. All the sub- Biology family 1 toxins potently block BK channels (e.g., of the The biological role of these K’ channel toxins is unknown slowpoke type), while the subfamily 2 and subfamily 3 tox- in detail, but they certainly contribute to the ins block only Kv channels (e.g., Shakerand Kvl.3). Within of the venoms. Though the toxins have been conserved subfamily 1, a-KTxl .l and a-KTxl.2 are broad specificity since the split between new and old world scorpions, they toxins that block K’ channels in both families (MacKinnon are always found as minor components (0.01 O/o-0.5%) of et al., 1988; Lucchesi et al., 1989) while a-KTxl.3 and Neuron 6

Table 1. Sequences of a-KTx Family Peptides

AL a -12 Turn Bi Commonname 0 5 10 15 20 25 30 35 a-KTx Name Reference Charybdotoxin-type: subfamily 1 CTx ZFTNVSCTTSKE-CWSVCQRLHNTSRG-KCMNKKCRCYS 1.1 Gimenez-Gallego et al., 1966 42 ZFTQESCTASNQ-CWSICKRLHNTNRG-KCMNKKCRCYS-- 1.2 Lucchesi et al., 1989 IbTx ZFTDVDCSVSKE-CWSVCKDLFGVDRG-KCMGKKCRCYQ 1.3 Galvez et al., 1990 LbTx VFIDVSCSVSKE-CWAPCKAAVGTDRG-KCMGKKCKCY?- - 1.4 Garcia-Calvo et al., 1993 Noxrustoxin-type: subfamrly 2 NTx TIINVKCT-SPKQCSKPCKELYGSSAGAKCMNGKCKCYNN 2.1 Possani et al., 1962 MgTx TI INVKCT-SPKQCLPPCKAQFGQSAGAKCMNGKCKCYP 2.2 Garcia-Calve et al., 1993 ClTxl ITINVKCT-SPQQCLRPCKDRFGQHAGGKCINGKCKCYP- - -- 2.3 Martin et al., 1994 Kaliotoxin-type: subfamily 3 KlTx GVE I NVKCSGSP-QCLKPCKDA-GMRFG-KCMNRKCHCTP? 3.1 Crest et al., 1992 AgTx2 GVPINVSCTGSP-QCIKPCKDA-GMRFG-KCMNRKCHCTPK 3.2 Garcia et al., 1994 AgTx3 GVPINVPCTGSP-QCIKPCKDA-GMRFG-KCMNRKCHCTPK-- 3.3 Garcia et al., 1994 AgTxl GVPINVKCTGSP-QCLKPCKDA-GMRFG-KCINGKCHCTPK 3.4 Garcia et al., 1994 KITx2 VRIPVSCKHSG-QCLKPCKDA-GMRFG-KCMNGKCDCTPK- - 3.5 Laraba-Djebari et al., 1994 Tytiustoxin-type: subfamily 4? TyKu VFINAKCRGSPE-CLPKCKEAIGKAAG-KCMNGKCKCYP- - - - 4.1 Rogowskr et al., 1994 Sequences of charybdotoxin rsoforms that have been characterized to date. Abbreviation for common names are shown, along with proposed formal names, according to a-KTx nomenclature. a-KTxl.4 are apparently specific for the BK variety (Galvez miniglobular proteins. All other side chains are surface- et al., 1990; Giangiacomo et al., 1993). exposed and project into aqueous solution. I wish to make one emphatic point about specificity. Reconstitution of single BK channels offers exceptional These toxins should not be considered pharmacological opportunities for examining toxin-channel interaction in tools that reliably dissect the numerous K+ currents found mechanistic detail. Most of our understanding of how the in neurons! We now have many examples of K+ channels peptide inhibits the channel comes from such studies on with similar functional behavior but huge differences in two toxins: a-KTxl .l and a-KTxl.3. All evidence points to sensitivities to toxin homologs, or, conversely, with large a simple bimolecular “plugging” mechanism, in which a functional differences but similar toxin affinities. In several single toxin molecule finds a specific site in the cases, we understand the molecular basis underlying external vestibule of the K+ channel and thereby occludes these varied toxin susceptibilities: slight sequence varia- the outer entry to the K’ conduction pore (Anderson et al., tions in the a-KTx receptor domain of the channels, differ- 1988; MacKinnon and Miller, 1988; Miller, 1988; Park and ences that have no consequences for channel gating be- Miller, 1992a; Giangiacomo et al., 1992). The demonstra- havior. For instance, alteration of only 2 residues in the tion that these toxins can be driven off the receptor site channel increases the binding affinity of a-KTxl .l five orders of magnitude (Goldstein and Miller, 1992); like- wise, the Kv2.1 channel, which is insensitive to a-KTx3.2, can be increased in sensitivity by at least six orders of magnitude by altering only 4 residues to those found in the Kvl.3 channel (Gross et al., 1994). Thus, the K’chan- nel field has avoided deluding itself into assigning channel genotype on the basis of pharmacological phenotype (a mistake, it seems to me, that is sometimes embraced en- thusiastically in the Ca2+channel field).

Toxin Structure and Mechanism of Block Multidimensional nuclear magnetic resonance methods have been used to determine the solution structuresof four of these toxins: a-KTxl .l (Bontems et al., 1991) a-KTxl.2 (Johnson and Sugg, 1992) a-KTx2.2 (Johnson et al., 1994) a-KTx3.1 (Fernandez et al., 1994) and a-KTx3.2 (Krezel et al., 1995). All show nearly identical polypeptide Figure 1. Polypeptide Backbone Structure of Charybdotoxin backbones consisting of a two- or three-turn a helix affixed Schematic representation of charybdotoxin backbone structure show- by bonds to a three-stranded 8 sheet (Figure 1). ing regions of a helix and 6 sheet. Side chains forming the center of the interaction surface are represented in red, and K27 is explicitly The three disulfide bonds maintain the peptide’s structural indicated. Disulfide bonds are shown in yellow. Coordinates deter- rigidity and wholly make up the internal volume of these mined with nuclear magnetic resonance are from Bontems et al., 1991. Review: Charybdotoxin Family of K’ Channel Blockers

l

specificially by K’ ions coming through the pore from the opposite side is a particularly strong indicator of the close apposition of the a-KTx receptor and the ion-selective pore, This blocking mechanism, initially worked out with single BK channels, was recently confirmed for Kv chan- nels (Goldstein and Miller, 1993). The entire molecular surface of one of these toxins, a-KTxl .l, has been functionally mapped by mutagenesis to identify that part of the toxin making direct contact with the K+ channel (Park and Miller, 1992a; Stampe et al., 1994; Goldstein et al., 1994). A synthetic gene for the toxin was expressed in bacteria, multiple point mutants were made at each solvent-exposed position, and each of these - 100 toxin variants was tested on both bilayer- reconstituted, Ca2+-activated K+ channels and oocyte- expressed, Shaker-type K+ channels, A clear picture of the a-KTx interaction surface emerged from this work (Figure 2A). The interaction surface for Ca’+-activated K’channels is composed of 8 residues that are contiguous in space (but not in sequence); for the Shaker channel, the interac- tion surface is slightly smaller, with 5 of these residues involved. The most sensitive residues are all found in the conserved sequence that defines the a-KTx family of pep- tides: K27, M29, and N30. By far the most important of these is K27; for example, in the Shakerchannel, a conser- vative mutation of this lysine to destabilizes the toxin over 1 OOO-fold. These crucial residues all project side chains off the second and third strands of the 5 sheet (Table 1, (j2 and (j3); most residues in the a helix project far away from the interaction surface, on the “back” side of the molecule. It is telling that among the extra residues in the larger interaction surface for the BK channel are F2 and W14, which are seen only in the toxins known to block BK channels, a-KTxl .n and a-KTxl.4. Besides being the residue most sensitive to mutation, K27 is unique in a palpably interpretable way (Park and Miller, 1992a, 1992b). This residue is known to project slightly into the K+ conduction pore, and hence must be located physically close to the central axis of the K+ chan- nel. This assertion is based on the fact that K27 mediates the repulsion between K’ ions in the pore and the bound Figure 2. Charybdotoxin Interaction Surface and Four-Fold Receptor toxin. As mentioned above, K+ ions in the pore destabilize Topography bound toxin. This is by itself not particularly surprising, (A) Space-filling model of charybdotoxin interaction surface (wrth since most a-KTx peptides carry a lot of positive charge, Shaker K’ channel), viewed normal to the pore axis. Red indicates which could interact electrostatically with K’ ions nearby strong sensitivity to conservative alterations, green represents indiffer- in the pore. What is both surprising and mechanistically ence to radical alteratron, and yellow labels residues with intermediate behavior. informative is that this interaction with K’ ions is wholly (B) Figure generated when the surface in (A) IS rotated in four-fold mediated by a single postively charged residue, K27. If symmetry about K27. K27 is neutralized, the destabilizing effect of K’ ions is lost completely; no other toxin residue has this property. Such a direct interaction between this one lysine group shape of the toxin’s three-dimensional footprint on the and K+ ions occupying binding sites within the conduction channel (Goldstein et al., 1994; Stampe et al., 1994; Hi- pore places K27 within a few angstroms of the channel’s dalgo and MacKinnon, 1995). Since the channel is four- four-fold axis of symmetry (Stampe et al., 1994). It is as fold symmetric around the central pore, it presents four though a-KTx family peptides have evolved to attack K+ equivalent orientations for a-KTx binding (MacKinnon, channels specifically by using the E amino group of K27 1991). Theshape of this receptor is assumed to be comple- as a chemically achievable approximation to a “tethered mentary to the shape generated when the interaction sur- K’ ion.” face (Figure 28) is stamped out four times around an ap- The localization of K27 permits a rough picture of the propriate axis. With this axis on K27, the a-KTx receptor Neuron 8

emerges as a rather flat plateau 25 x 35 A wide, sur- rounded by a low (lo-15 A) wall at the outer end of EAGSENSF,,,FK,,,SIPD,,,* * * * * * MT,,PVGVWGK the narrow pore. Geometrical considerations demand that toxin bound to its receptor touches all four channel sub- units simultaneously and eccentrically (Stampe et al., 1994; Hidalgo and MacKinnon, 1995). Though a-KTxl.l is the only toxin that has been func- tionally mapped in this way, it is likely that the conclusions drawn for this toxin will apply broadly to the others. In -. particular, the sequence and structural similarities, along with the common mechanism of action, argue that all a-KTx homologs will expose interaction-surface residues off the 6 sheet, with K27 making intimate contact with the central pore.

The a-KTx Receptor and the K+ Channel Vestibule A major motivation for examining a-KTx peptides in such detail is the expectation that, by virtue of their known struc- tures, the toxins might tell us something about the K+chan- nel, whose structure is, of course, understood only in the vaguest terms. This expectation is being fulfilled; these toxins have indeed revealed structural and molecular fea- Figure 3. a-KTX Receptor Sequence in Voltage-Gated K* Channels tures of these channels. The identification, by conven- The a-KTX receptor determinants in voltage-gated K’ channels are tional mutagenesis of the Shaker channel, of negatively shown in the context of the molecular architecture of these channels. charged residues contributing to the binding energy of All known receptor determinants are found on the two sides of the selectivity-determining region (P). The a-KTX receptor sequence of a-KTx peptides(MacKinnon and Miller, 1989) immediately the Shaker K’channel is shown, with toxin-mapped residues indicated. pointed to the region of channel sequence likely to be Solid bar indicates the charybdotoxin “footprint’in the linear sequence. carrying pore-lining residues, which had not at that time Residues with numbered positions are those that have been located been found for voltage-gated ion channels. As a result, in three-dimensional space by toxin-receptor comutagenesis. the P region of voltage-gated K+ channels was quickly found by several groups (Yellen et al., 1991; Hartmann et al., 1991). This stretch of about 40 residues lying between the pore. This footprint is located precisely where, in the S5 and S6 in voltage-gated K+ channels (Figure 3) is now absence of any direct molecular information, the pore- known to contain all the a-KTx receptor determinants blocking mechanism had predicted that it should be (Park (MacKinnon et al., 1990; Goldstein et al., 1994) as well and Miller, 1992b); this harmony between mechanistic ex- as the pore-lining residues most crucial for ion selectivity pectation and molecular fact is a powerful corroboration (Kirsch et al., 1992; de Biasi et al., 1993; Heginbotham et of the direct pore-plugging mechanism of a-KTx action. al., 1993). After locating on both toxin and channel the residues Besides pointing to the pore-forming sequences of K+ involved in binding, the next step has been to match these channels, analysis of a-KTx block showed that these up, identifying pairs of residues, one each on channel and channels are tetrameric, a result expected but not toxin, that interact in the bound complex. At this writing, demonstrated. Using a Shaker point mutant that disrupts four such interaction partners have been located. Such a-KTx3.2 binding in a recessive fashion, MacKinnon (1991) partners are indicated from the energetics of binding when showed that the channel presents four energetically equiv- four sets of mutants are examined. For example, when alent orientations for toxin to bind, as expected for a four- Shaker carries a small residue at position 425, then fold symmetric tetramer. a-KTxl .l binds equally well, regardless of the size of the By performing scanning mutagenesis on the P region residues at positions 8 and 9; however, if the channel is of the Shaker K+ channel, MacKinnon et al. (1990) identi- given a large residue at this position, binding becomes fied the residues affecting a-KTx3.2 block, and these were sensitive to the size of the residues at these positions on confirmed for a-KTxl .l (Goldstein et al., 1994). These resi- the toxin (Goldstein et al., 1994). This argues that the dues lie in the two wings of sequence flanking the strongly “bump” formed by positions 8 and 9 on the toxin surface conserved P region hairpin (Figure 3). Residues N-terminal makes steric contact with a large residue at position 425; to position 425 and C-terminal to position 451 influence this contact, in combination with the known structure of a-KTx block only weakly, probably by through-space elec- the toxin and the central location of K27, places this Shaker trostatic effects (MacKinnon and Miller, 1989; MacKinnon residue at a fixed position in three-dimensional space with et al., 1990; Goldstein et al., 1994); strong influences, man- respect to the pore axis. Variations on this method using ifested in greatly altered dissociation rates of toxin, appear electrostatic interactions between residue pairs (Stocker abruptly between these positions, as if reflecting the tox- and Miller, 1994), and a thermodynamic cycle analysis of in’s one-dimensional footprint covering the outer part of such mutant pairs (Hidalgo and MacKinnon, 1995), have Review: Charybdotoxin Family of K’ Channel Blockers 9

Chrcchi, G. G, Grmenez-Gallego, G., Ber, E., Garcia, M. L., Winquist, R., and Cascieri, M. A. (1988). Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebreus venom. J. Biol. Chem. 263, 10192-10197. Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M. (1992). Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca-activated K channels charac- terized from Androctonus mauretanicus mauretanicus venom. J. Biol. Chem. 267, 1640-1647. de Biasi. M., Drewe, J. A., Kirsch, G. E., and Brown, A. M. (1993). substituion identifies a surface position and confers Cs+ se- lectivity on a K’ pore. Biophys. J. 65, 1235-1242. Fernandez, I., Romi, R., Szendeffy, S., Martin-Eauclaire, M. F., Ro- chat, H., Van Rietschoten, J., Ports, M., and Giralt, E. (1994). Kaliotoxin Figure 4. Spatial Locations of Specific Residues rn the Shaker Ves- (l-37) shows structural differences with related tibule blockers. Biochemistry 33, 14256-14263. Approximate positions of 4 channel residues, localized by comutagen- Galvez, A., Gimenez-Gallego, G., Reuben, J. P., Roy-Contancin, L., esis, are shown on a Shaker K’ channel subunit. Feigenbaum, P., Kaczorowski, G. J., and Garcia, M. L. (1990). Purifica- tion and characterization of a unrque, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom estimated in addition the locations of Shaker residues 427, of the scorpion Buthus tamulus. J. Biol. Chem. 265, 11083-l 1090. 431, and 449 (Figure 4). This cartoon of the outer vestibule Garcia,M. L., Garcia-Calve,M., Hidalgo,P., Lee,A., and MacKinnon, of the Shaker K+ channel places specific residues in the R. (1994). Purification and characterization of three inhibitors of volt- P region on the topographic map of the vestibule deduced age-dependent K+ channels from Leiurus quinquestriatus var. hebreus venom. Biochemistry 33, 6834-6839. from four-fold rotation of the complementary shape of the Garcia-Calvo, M., Leonard, R. J., Novick, J., Stevens, S. P., Schmal- a-KTxl .l interaction surface about K27. hofer, W., Kaczorowski, G. J., and Garcia, M. L. (1993). Purification, characterizatron, and biosynthesis of , a component of Cen- Conclusion truroides mararitatus venom that selectively inhibits voltage- The a-KTx family of K’ channel blockers is providing a dependent potassium channels. J. Biol. Chem. 268, 18866-18874. rich collection of informative molecular probes for the K’ Garcia-Calvo, M., Knauss, H. G., McManus, 0. B., Giangiacomo, K. M., Kaczorowski, G. J., and Garcia, M. L. (1994). Purification and channels. Moreover, they have given us the first glimpse reconstitution of the high-conductance calcium-activated potassium of the size and shape of a mechanistically interesting part channel from tracheal smooth muscle. J. Biol. Chem. 269, 676-682. of K+ channels: the outer opening of the ion conduction Giangiacomo, K. M., Garcia, M. L., and McManus, 0.6. (1992). Mecha- pore. They are just beginning to facilitate protein-level bio- nism of iberiatoxin block of the large-conductance calcium-activated chemical approaches to these channels as well (Vazquez potassium channel from bovine aortic smooth muscle. Biochemistry et al., 1989; Knaus et al., 1994; Garcia-Calvo et al., 1994; 37, 6719-6727. Sun et al., 1994) since they bind only to the fully assem- Giangiacomo, K. M., Sugg, E. E., Garcia-Calvo, M., Leonard, R. J., McManus, 0. E., Kaczorowski, G. J., and Garcia, M. L. (1993). Syn- bled tetrameric channel. We can expect that many more theticcharybdotoxin- chimericpeptidesdefine toxin binding such toxins, with unexpected specificites, will be de- sites on calcium-activated and voltage-dependent potassium chan- scribed in the coming years, since many scorpion venoms nels. Biochemistry 32, 2363-2370. remain uninvestigated. These peptides have found utility Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczor- at the intersection of several lines of molecular neurobiol- owski, G. J., and Garcia, M. L. (1988). Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of cal- ogy: pharmacology, biochemistry, and structure-function mum-activated potassium channels, Proc. Natl. Acad. Sci. USA 85, analysis of K+ channels. 3329-3333. Goldstein, S. A. N., and Miller, C. (1992). A point mutation in a Shaker June 14. 1995 K’ channel changes its charybdotoxin binding site from low to high affinity. Biophys. J. 62, 5-7. References Goldstein, S. A. N., and Miller, C. (1993). Mechanism of charybdotoxin block of a Shaker K+ channel. Biophys. J. 65, 1613-1619. Anderson, C., MacKinnon, R., Smith, C., and Miller, C. (1988). Cha- rybdotoxin inhibition of Ca2‘-activated K’channels. Effects of channel Goldstein, S. A. N., Pheasant, D. J., and Miller, C. (1994). The cha- gating, voltage, and ionic strength. J. Gen. Physiol. 91, 317-333. rybdotoxin receptor of a Shaker K‘ channel: peptide and channel resi- dues mediating molecular recognition. Neuron 72, 1377-1388. Bontems, F., Roumestand, C., Gilquin, B., Menez, A., and Toma, F. (1991). Refined structure of charybdotoxin: common motifs in scorpion Gross, A., Abramson, T., and MacKinnon, R. (1994). Transfer of scor- toxins and insect defensins. Science 254, 1521-1523. pion toxin receptor to an insensitive potassium channel. Neuron 13, 961-966. Carbone, E., Wanke, E., Prestipino, G., Possani, L. D., and Maelicke, A. (1982). Selective blockage of voltage-dependent K’ channels by a Hartmann, H. A., Kirsch, G. E., Drewe, J. A., Taglialatela, M., Joho, novel scorpion toxin. Nature 296, 90-91. R. H., and Brown, A. M. (1991). Exchange of conduction pathways between two related K+ channels. Science 257, 942-944. Catterall, W. A. (1977). Membrane potential-dependent binding of scor- pion toxin to the action potential of Na+ ionophore. Studies with a toxin Heginbotham, L., Lu, Z., Abramson, T., and MacKinnon, R. (1993). derivative prepared by lactoperoxidase-catalyzed iodination. J. Biol. Mutations in the K’ channel signature sequence. Biophys. J. 66,1061- Chem. 252, 8660-8688. 1067. Catterall, W. A. (1980). that act on voltage-sensitive so- Hidalgo, P., and MacKinnon, R. (1995). Revealing the architecture of dium channels in excitable membranes. Annu. Rev. Pharmacol. Tox- a K’ channel pore through mutant cycles with a peptide inhibitor. Sci- icol. 20. 15-43. ence 268, 307-310. Neuron 10

Hughes, M., Romey, G., Duval, D., Vincent, J. P., and Lazdunski, Rogowski, R. S., Krueger, B. K., Collins, J. H., and Blaustein, M. P. M. (1982). Apamin as a selective blocker of the calcium dependent (1994). Tityustoxin Ka blocks voltage-gated noninactivating K’ than potassium channel in neuroblastoma cells: voltage-clamp and bio- nels and unblocks inactivating K’ channels blocked by a-dendrotoxin chemical characterization of the toxin receptor. Proc. Natl. Acad. Sci. in synaptosomes. Proc. Natl. Acad. Sci. USA 97, 1475-1479. USA 79, 1308-1312. Stampe, P., Kolmakova-Partensky, L., and Miller, C. (1994). Intima- Hurst, R. S., Busch, A. E., Kavanaugh, M. P., Osborne, P. B., North, tions of K’ channel structure from a complete functional map of the R. A., and Adelman, J. P. (1991). Identification of residues molecular surface of charybdotoxin. Biochemistry 33, 443-450. mvolved in dendrotoxin block of rat voltage-dependent potassium Stocker, M., and Miller, C. (1994). Electrostatic distance geometry in channels. Mol. Pharmacol. 40, 572-576. a K’ channels vestibule. Proc. Natl. Acad. Sci. USA 97, 9509-9513. Johnson, B. A., and Sugg, E. E. (1992). Determination of the three- Sun, T., Naini, A. A., and Miller, C. (1994). High-level expression and dimensional structure of iberiatoxin in solution by ‘H nuclear magnetic functional reconstitution of Shaker K’ channels. Biochemistry 33, resonance spectroscopy. Biochemistry 37, 8151-8159. 9992-9999. Johnson, B. A., Stevens, S. P., and Williamson, J. M. (1994). Determi- Vazquez. J., Feigenbaum, P., Katz, G., King, V. F., Reuben, J. P.. nation of the three-dimensional structure of margatoxin by ‘H, ‘C, ‘5N Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, triple-resonance nuclear magnetic resonance spectroscopy. Biochem- M. L. (1989). Characterization of high affinity binding sites for chary- istry 33, 15061-l 5070. bdotoxin in sarcolemmal membranes from bovine aortic smooth mus- Kirsch, G. E., Drewe, J. A., Hartmann, H. A., Taglialatela, M., de Biasr, cle. Evidence for a direct association with the high conductance cal- M., Brown, A. M., and Joho, Ft. H. (1992). Differences between the cium-activated potassium channel. J. Biol. Chem. 264,20902-20909. deep pores Of K+ channels determined by an interacting pair of nonpo- Yellen, G.. Jurman, M., Abramson, T., and MacKinnon, R. (1991). lar amino acids. Neuron 8, 499-505. Mutatrons affecting internal TEA blockade identify the probable pore- Knaus, H. G., Garcia-Calve, M., Kaczorowski, G. J., and Garcia, forming region of a K’ channel. Science 257, 939-942. M. L. (1994). Subunit composition of the high conductance calcium- activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels. J. Biol. Chem. 269, 3921-3924. Krezel, A., Kasibhatla, C., Hidalgo, P., MacKinnon. R., and Wagner, G. (1995). Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry. Protein Sci., in press. Laraba-Djebari, F., Legros, C., Crest, M., Ceard, B., Romi, R., Man- suelle, P., Jacquet, G., Van Rietschoten, J., Gola, M., Rochat, H.. Bougrs, P. E., and Martin-Eauclaire, M. F. (1994). The kaliotoxin family enlarged. J. Biol. Chem. 269, 32835-32843. Lucchesi, K., Ravindran, A., Young, H., and Moczydlowski, E. (1989). Analysis of the blocking activity of charybdotoxin homologs and iodin- ated derivatives against Ca2+-activated K’ channels. J. Membr. Biol. 109, 269-281. MacKinnon, R. (1991). Determination of the subunit stoichiometry of a voltage-activated potassium channel. Nature 500, 232-235. MacKinnon, R., and Miller, C. (1988). Mechanism of charybdotoxin block of Ca”-activated K’ channels, J. Gen. Physiol. 97, 335-349. MacKinnon, R., and Miller, C. (1989). Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor. Science 245, 1382-1385. MacKinnon, R., Reinhart, P. H., and White, M. M. (1988). Charybdo- toxin block of Shaker K+ channels suggests that different types of K’ channels share common structural features. Neuron 1, 997-1001. MacKinnon, R., Heginbotham, L., and Abramson, T. (1990). Mapping the receptor site for charybdotoxin, a pore-blocking potassium channel inhibitor. Neuron 5, 767-771. Martin, B. M., Ramirez, A. N., Gurrola, G. 6.. Nobtle, M., Prestipino, G., and Possani, L. (1994). Novel K’channel-blocking toxins from the venom of the scorpion Cenfruroides limpidus limpidus Karsch. Bio- them. J. 304, 51-56. Miller, C. (1988). Competition for block of a Ca*+-activated K’channel by charybdotoxin and . Neuron 1. 1003-1006. Parcej, D. N., and Dolly, J. 0. (1989). Dendrotoxin acceptor from bo- vine synaptic plasma membranes. Binding properties, purification and subunit composition of a putative constituent of certain voltage- activated K’ channels. Biochem. J. 257, 899-903. Park, C.-S., and Miller, C. (1992a). Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel. Neuron 9, 307-313. Park, C.-S., and Miller, C. (1992b). Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin. Biochemistry 37, 7749-7755. Possani, L. D., Martin, B. M., and Svendensen, I. (1982). The primary structure of noxiustoxin: a K channel blocking peptide, purified from the venom of the scorpion Cenfruroides noxius hoffmann. Calsberg Res. Commun. 47, 285-289.