Gating Modifier Toxins Research Articles

Non-Toxin Gating Modifiers Reveal the Promiscuous Nature of the Voltage Sensor of Kv7.2 and TRPV1 Channels

Voltage-sensitive cation channel architecture consists of two main structural modules, the voltage sensor domain (VSD) and the pore domain. The VSDs are the target of various gating-modifier toxins and their paddle motifs are modular and transferable structures. Here we show that NH17 and NH29, two new Kv7.2 channel blocker and opener, respectively, act as non-toxin gating modifiers. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2 and S4 in a way, which stabilizes the interaction between two conserved charged residues in S2, and S4, known to interact electrostatically, in the open state of Kv channels. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that described for tarantula toxins. TRPV1 channels appear to be weakly voltage-dependent. However, NH29 changes the linear TRPV1 current-voltage relation obtained with capsaicin, to an outwardly rectifying shape. Interestingly, mutations in linker S3-S4 of the TRPV1 VSD are significantly more resistant to the inhibitory effect of NH29. While compound NH17 potently blocks Kv7.2 channels, it sensitizes the TRPV1 current activated by capsaicin. Mutations in linker S3-S4 switch the TRPV1 sensitizing action of NH17 to a potent inhibition. Subtle modifications in the VSD or in the chemical structure of the molecule drastically change the attributes of the gating-modifier, thereby stabilizing the channel in either the closed or the open state. Data indicate that the novel compounds may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea anemone toxins. Thus, the VSDs of Kv and TRPV1 channels are promiscuous and share some common structural and biophysical features.

Molecular Dynamics Simulations of Kv Channels and Gating-Modifier Peptide Toxins

Activation of voltage-dependent K+ (Kv) channels modulates cellular excitability in several physiological systems including neuronal and cardiac cells. Given recent progress in structure determination and in computation technology, molecular dynamics (MD) simulation analyses have become a valuable tool in studies of voltage-dependent gating of Kv channels. However, due to the complex voltage-sensing mechanisms and interplays between the voltage sensor and the ion-conduction pathway, and because of limitations in MD simulation technology, the technical details are crucial to performing meaningful simulations. This review briefly introduces the current understanding of Kv channel molecular physiology, and then considers, in depth, the progress in Kv channel simulations and the physiological insights on Kv channels obtained from such computational studies. Effectiveness of several techniques involving steered molecular dynamics, potential mean force analyses and coarse-grained simulation methods in the analyses of interactions among helices of the voltage sensor domain (VSD) and the coupling between VSD and the pore domain is stressed. The interactions among helices of the VSD, which determine the kinetics of the voltage-dependent gating, are strong, and therefore the potential usefulness of artificial restraints is discussed. Given information of an extensive set of mutants that effect gating kinetics, usefulness of in silico mutagenesis is stressed. Progress in??molecular dynamics simulation analyses of gating modifier toxins is also reviewed. Based on the two different binding modes observed in the MD simulation, a model is proposed for the mechanisms of these gating modifiers. This model gives us insight on some of the enigmas associated with the gating modifiers involving hanatoxin, VsTx1 and GsMTx4.

S3b Amino Acid Substitutions and Ancillary Subunits Alter the Affinity of Heteropoda venatoria Toxin 2 for Kv4.3

Heteropoda venatoria toxin 2 (HpTx2) is an inhibitor cystine knot (ICK)-gating modifier toxin that selectively inhibits Kv4 channels. To characterize the molecular determinants of interaction, we performed alanine scanning of the Kv4.3 S3b region. HpTx2-Kv4.3 interaction had an apparent K(d) value of 2.3 microM. Two alanine mutants in Kv4.3 increased K(d) values to 6.4 microM for V276A and 25 microM for L275A. Simultaneous mutation of both amino acids to alanine nearly eliminated toxin interaction. Unlike Hanatoxin and other well characterized ICK toxins, HpTx2 binding does not require a charged amino acid for interaction. To determine whether the identity of the S3b binding site amino acids altered HpTx2 specificity, we constructed Kv4.3 [LV275IF]. This mutation decreased the K(d) value to 0.54 microM, suggesting that the hydrophobic character of the putative binding site is the most important property for interaction with HpTx2. One mutant, N280A, caused stronger interaction of HpTx2 with Kv4.3; the K(d) value for Kv4.3 [N280A] was 0.26 microM. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 coexpressed with KChIP2b. The toxin's K(d) value for Kv4.3 + KChIP2b was 0.95 microM. KChIP2b stabilizes the closed state of Kv4.3, suggesting that the increased toxin affinity is due to increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins but that the interventions that increase toxin affinity suggest flexibility toward channel binding that belies its unusual specificity for Kv4 channels.

HpTx2 Interaction With Kv4.3 and Kv4.1 Reveals Differences in Gating Modification

HpTx2 is an ICK gating modifier toxin specific for Kv4 channels. The Kd of HpTx2 for two similar channels, Kv4.3 and Kv4.1, was 2.4 μM and 7.1 μM, respectively. HpTx2 inhibition of Kv4.3 is highly voltage dependent, shifting the current-voltage relationship to more depolarized potentials; consistent with the classic behavior of gating modifier toxins. However, modification of Kv4.1 gating is much less voltage dependent. Site directed mutagenesis of the S3b interaction site of Kv4.3 and Kv4.1 shows that the same two conserved bulky hydrophobic amino acids are required for HpTx2 interaction with each channel. While the interior of the binding site is conserved between Kv4.3 and Kv4.1, three amino acids adjacent to the binding site are not conserved. Swapping these amino acids between Kv4.3 and Kv4.1 swaps the phenotypic response to toxin, while having minimal effect on gating properties of the channels. We modeled the activation gating of Kv4.3 and Kv4.1 and incorporated the effects of HpTx2 into the kinetic parameters of activation. The model is similar for both channels; it has four voltage-dependent transitions between the closed states followed by voltage-independent transition to an open state. Voltage-dependent transitions in Kv4.3 are more strongly affected by toxin. In Kv4.1, the voltage-independent transition from the closed pre-open to open state is most affected by HpTx2. Therefore, a higher proportion of toxin-bound Kv4.1 channels are in the closed pre-open state, compared to toxin-bound Kv4.3 channels. This decreases the voltage dependence of toxin-bound Kv4.1 opening. The model closely recapitulates our experimental data. These data show that amino acids near the HpTx2 binding site play a role in the kinetics of Kv4 channel activation gating.

Charge-dependent And Isoform-specific Interactions Between ProTxii And T-type Calcium Channels

ProTxII, peptide toxin isolated from the venom of the tarantula Thrixopelma puriens, modifies voltage-dependent activation of both T-type calcium (Ca) channels and voltage-gated sodium (Na) channels. In the presence of ProTxII (5μM) the voltage at half maximal activation (V1/2) of the CaV3.1 isoform is shifted positive (>25mV) and maximum conductance (Gmax) decreases (∼50%). Interestingly, the toxin's effects on this channel were completely precluded in the presence of high extracellular divalent concentrations indicating a role for surface charge-like, electrostatic interactions with the channel. Several mutant toxins in which individual basic residues were neutralized were tested for activity on CaV3.1. Three of these mutants, R13Q, R22A, and K28A, significantly disrupted the ability of the toxin to both shift channel activation and decrease Gmax. Two other mutations: K4Q and K14A, showed minimal or no effect, thus indicating an important yet specific role of charge in ProTxII's interaction with CaV3.1. The gating kinetics of T-type Ca channels varies among the three known isoforms suggesting there might be differences in the gating structures and, therefore, potential gating modifier toxin interaction surfaces as well. In CaV3.3, 5μM ProTxII reduced Gmax by approximately 60%, similar to what was seen for CaV3.1. However, unlike in CaV3.1, this concentration of toxin produced only a minimal shift in voltage dependent activation (2mV). These results suggest significant differences in the extracellular surface of T-type Ca channels across isoforms, particularly in terms of surface charge distribution close to one or more of the channels’ voltage sensors.

Ancillary subunits and S3b amino acid substitutions alter the affinity of HpTx2 for Kv4.3

HpTx2 is an ICK gating modifier toxin that selectively inhibits Kv4 channels, making it an excellent probe for understanding Kv4 structure and function. To characterize the molecular determinants of interaction, we performed alanine scanning of Kv4.3 S3b linker. HpTx2-Kv4.3 interaction had a Kd = 2.4 μM. Two alanine mutants in Kv4.3 caused a dramatic increase in Kd: 6.4 μM for V276A and 25 μM for L275A. LV275AA nearly eliminated toxin interaction. Unlike HaTx and other well-characterized ICK toxins, HpTx2 binding does not require a charged amino acid. To determine if the identity of the amino acids altered HpTx2 specificity, we constructed LV275IF. This mutation decreased the Kd to 0.54 μM, suggesting that the hydrophobic character of the binding site is the most important property for interaction with HpTx2. Two of the alanine mutants, N280A and D282A caused stronger interaction of HpTx2 with Kv4.3; the Kd for Kv4.3 [N280A] was 0.26 μM. We propose that these mutations either remove a steric barrier to HpTx2 occupation of S3b, or that removal of these polar side chains increases toxin affinity by increasing the hydrophobic character of the binding site. To understand Kv4.3-based transient outward currents in native tissues, we tested the affinity of HpTx2 for Kv4.3 co-expressed with KChIP2b. The toxin's Kd for Kv4.3+KChIP2b was 0.95 μM. HpTx2 binds to the closed state of Kv4.3, which is stabilized by KChIP2b. We propose the increased affinity is due to the increased stabilization of the closed state. These data show that HpTx2 binding to Kv4.3 has aspects in common with other ICK gating modifier toxins, but that the interventions that increase toxin affinity suggests flexibility toward channel binding that belies its unusual specificity for Kv4 channels.

Lipid Bilayer Deformation and the Free Energy of Interaction of a Kv Channel Gating-Modifier Toxin

A number of membrane proteins act via binding at the water/lipid bilayer interface. An important example of such proteins is provided by the gating-modifier toxins that act on voltage-gated potassium (Kv) channels. They are thought to partition to the headgroup region of lipid bilayers, and so provide a good system for probing the nature of interactions of a protein with the water/bilayer interface. We used coarse-grained molecular dynamics simulations to compute the one-dimensional potential of mean force (i.e., free energy) profile that governs the interaction between a Kv channel gating-modifier toxin (VSTx1) and model phospholipid bilayers. The reaction coordinate sampled corresponds to the position of the toxin along the bilayer normal. The course-grained representation of the protein and lipids enabled us to explore extended time periods, revealing aspects of toxin/bilayer dynamics and energetics that would be difficult to observe on the timescales currently afforded by atomistic molecular dynamics simulations. In particular, we show for this model system that the bilayer deforms as it interacts with the toxin, and that such deformations perturb the free energy profile. Bilayer deformation therefore adds an additional layer of complexity to be addressed in investigations of membrane/protein systems. In particular, one should allow for local deformations that may arise due to the spatial array of charged and hydrophobic elements of an interfacially located membrane protein.

Evidence for multiple effects of ProTxII on activation gating in Na V1.5

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na + and Ca 2+ channels. ProTxII is distinct from other known Na + channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na V1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G max appeared at relatively low concentrations (40–100 nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (≥500 nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba 2+, known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba 2+ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.

Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao.

Cystine knot toxins (CKTs) in spider venoms represent a rich source of novel ligands for varied ion channels. Here, we identified 95 novel putative CKT precursors by analyzing expressed sequence tags of the tarantula Chilobrachys jingzhao venom gland. Phylogenetics analyses revealed one orphan family and six families with sequence similarity to known toxins. To further investigate the relationships of their structures, functions and evolution, we assayed 10 representative toxins for their effect on ion channels, and performed structure model comparisons, evolution analysis and toxin distribution analysis. This study revealed two major types of CKTs: pore-blocking toxins and gating modifier toxins. A few blockers were observed with relatively high abundance and wide distribution, which may be a category of original toxins that block channels conserved in various preys with relatively high specificity. The gating modifier families contain advanced toxins, usually have many members and interact with diverse regulatory components of channels.

Inhibition of Sodium Channel Gating by Trapping the Domain II Voltage Sensor with Protoxin II

ProTx-II, an inhibitory cysteine knot toxin from the tarantula Thrixopelma pruriens, inhibits voltage-gated sodium channels. Using the cut-open oocyte preparation for electrophysiological recording, we show here that ProTx-II impedes movement of the gating charges of the sodium channel voltage sensors and reduces maximum activation of sodium conductance. At a concentration of 1 microM, the toxin inhibits 65.3 +/- 4.1% of the sodium conductance and 24.6 +/- 6.8% of the gating current of brain Na(v)1.2a channels, with a specific effect on rapidly moving gating charge. Strong positive prepulses can reverse the inhibitory effect of ProTx-II, indicating voltage-dependent dissociation of the toxin. Voltage-dependent reversal of the ProTx-II effect is more rapid for cardiac Na(v)1.5 channels, suggesting subtype-specific action of this toxin. Voltage-dependent binding and block of gating current are hallmarks of gating modifier toxins, which act by binding to the extracellular end of the S4 voltage sensors of ion channels. The mutation L833C in the S3-S4 linker in domain II reduces affinity for ProTx-II, and mutation of the outermost two gating-charge-carrying arginine residues in the IIS4 voltage sensor to glutamine abolishes voltage-dependent reversal of toxin action and toxin block of gating current. Our results support a voltage-sensor-trapping model for ProTx-II action in which the bound toxin impedes the normal outward gating movement of the IIS4 transmembrane segment, traps the domain II voltage sensor module in its resting state, and thereby inhibits channel activation.

β-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the β-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of β-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

APETx1 from Sea Anemone Anthopleura elegantissima Is a Gating Modifier Peptide Toxin of the Human Ether-a-go-go- Related Potassium Channel

We studied the mechanism of action and the binding site of APETx1, a peptide toxin purified from sea anemone, on the human ether-a-go-go-related gene (hERG) channel. Similar to the effects of gating modifier toxins (hanatoxin and SGTx) on the voltage-gated potassium (Kv) 2.1 channel, APETx1 shifts the voltage-dependence of hERG activation in the positive direction and suppresses its current amplitudes elicited by strong depolarizing pulses that maximally activate the channels. The APETx1 binding site is distinctly different from that of a pore-blocking peptide toxin, BeKm-1. Mutations in the S3b region of hERG have dramatic impact on the responsiveness to APETx1: G514C potentiates whereas E518C abolishes the APETx1 effect. Restoring the negative charge at position 518 (methanethiosulfonate ethylsulfonate modification of 518C) partially restores APETx1 responsiveness, supporting an electrostatic interaction between E518 and APETx1. Among the three hERG isoforms, hERG1 and hERG3 are equally responsive to APETx1, whereas hERG2 is insensitive. The key feature seems to be an arginine residue uniquely present at the 514-equivalent position in hERG2, where the other two isoforms possess a glycine. Our data show that APETx1 is a gating modifier toxin of the hERG channel, and its binding site shares characteristics with those of gating modifier toxin binding sites on other Kv channels.

Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5: IMPLIED EXISTENCE OF A NOVEL TOXIN BINDING SITE COUPLED TO ACTIVATION

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.

SGTx1, a Kv Channel Gating-Modifier Toxin, Binds to the Interfacial Region of Lipid Bilayers

SGTx1 is a gating-modifier toxin that has been shown to inhibit the voltage-gated potassium channel Kv2.1. SGTx1 is thought to bind to the S3b-S4a region of the voltage-sensor, and is believed to alter the energetics of gating. Gating-modifier toxins such as SGTx1 are of interest as they can be used to probe the structure and dynamics of their target channels. Although there are experimental data for SGTx1, its interaction with lipid bilayer membranes remains to be characterized. We performed atomistic and coarse-grained molecular dynamics simulations to study the interaction of SGTx1 with a POPC and a 3:1 POPE/POPG lipid bilayer membrane. We reveal the preferential partitioning of SGTx1 into the water/membrane interface of the bilayer. We also show that electrostatic interactions between the charged residues of SGTx1 and the lipid headgroups play an important role in stabilizing SGTx1 in a bilayer environment.

Solution Structure and Functional Characterization of Jingzhaotoxin-XI: A Novel Gating Modifier of both Potassium and Sodium Channels,

JZTX-XI is a peptide toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. It contains 34 residues including six cysteine residues with disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Using 3'- and 5'-RACE methods, the full-length cDNA was identified as encoding an 86-residue precursor of JZTX-XI. In the electrophysiological assay, JZTX-XI shows activity toward the Kv2.1 channel in a way similar to hanatoxin1 and SGTx1 that both the activation and the deactivation processes are affected, which is in accordance with the high sequence homology among them (over 60% identity). On the other hand, JZTX-XI also exhibits specific interaction against the Nav channels of rat cardiac myocytes with a significant reduction in the peak current and slowing of channel inactivation. The solution structure of native JZTX-XI was determined by 1H NMR methods to identify the structural basis of these specific activities. Structural comparison of JZTX-XI with other gating modifier toxins shows that they all adopt a similar surface profile, a hydrophobic patch surrounded by charged residues such as Arg or Lys, which might be a common structural factor responsible for toxin-channel interaction. JZTX-XI might be an ideal tool to further investigate how spider toxins recognize various ion channels as their targets.

Functional Interactions at the Interface between Voltage-Sensing and Pore Domains in the Shaker Kv Channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

Voltage-gated ion channels and gating modifier toxins

Voltage-gated sodium, calcium, and potassium channels generate electrical signals required for action potential generation and conduction and are the molecular targets for a broad range of potent neurotoxins. These channels are built on a common structural motif containing six transmembrane segments and a pore loop. Their pores are formed by the S5/S6 segments and the pore loop between them, and they are gated by bending of the S6 segments at a hinge glycine or proline residue. The voltage sensor domain consists of the S1–S4 segments, with positively charged residues in the S4 segment serving as gating charges. The diversity of toxin action on these channels is illustrated by sodium channels, which are the molecular targets for toxins that act at six or more distinct receptor sites on the channel protein. Both hydrophilic low molecular weight toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Hydrophobic alkaloid toxins and related lipid-soluble toxins act at intramembrane sites and alter voltage-dependent gating of sodium channels via an allosteric mechanism. In contrast, polypeptide toxins alter channel gating by voltage-sensor trapping through binding to extracellular receptor sites, and this toxin interaction has now been modeled at the atomic level for a β-scorpion toxin. The voltage-sensor trapping mechanism may be a common mode of action for polypeptide gating modifier toxins acting on all of the voltage-gated ion channels.

Gating modifier toxins of voltage-gated calcium channels

Some of the most potent and specific inhibitors of voltage-gated calcium channels are peptide toxins that inhibit channel function not by occlusion of the channel pore, but rather by interfering with the voltage dependence and kinetics of channel opening and closing. Many such gating modifier toxins conform to the inhibitor cystine knot structural family and have primary sequence or functional mechanism similar to toxins that target voltage-gated sodium or potassium channels. This review introduces known gating modifiers of calcium channels, discusses the selectivity, binding sites, and mechanism of the toxin–channel interaction, and reviews the usefulness of these toxins as research tools and as the basis for novel calcium channel pharmacology and therapeutics.

Binding of a Gating Modifier Toxin Induces Intersubunit Cooperativity Early in the Shaker K Channel's Activation Pathway

Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shaker's activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions.

Four Novel Tarantula Toxins as Selective Modulators of Voltage-Gated Sodium Channel Subtypes

Four novel peptide toxins that act on voltage-gated sodium channels have been isolated from tarantula venoms: ceratotoxins 1, 2, and 3 (CcoTx1, CcoTx2, and CcoTx3) from Ceratogyrus cornuatus and phrixotoxin 3 (PaurTx3) from Phrixotrichus auratus. The pharmacological profiles of these new toxins were characterized by electrophysiological measurements on six cloned voltage-gated sodium channel subtypes expressed in Xenopus laevis oocytes (Na(v)1.1/beta(1), Na(v)1.2/beta(1), Na(v)1.3/beta(1), Na(v)1.4/beta(1), Na(v)1.5/beta(1), and Na(v)1.8/beta(1)). These novel toxins modulate voltage-gated sodium channels with properties similar to those of typical gating-modifier toxins, both by causing a depolarizing shift in gating kinetics and by blocking the inward component of the sodium current. PaurTx3 is one of the most potent peptide modulators of voltage-gated sodium channels described thus far from spider venom, modulating Na(v)1.2 with an IC(50) value of 0.6 +/- 0.1 nM. CcoTx1 and CcoTx2, differing by only one amino acid, are potent modulators of different voltage-gated sodium channel subtypes from the central nervous system, except for Na(v)1.3, which is only affected by CcoTx2. The potency of CcoTx3 is lower, although this toxin seems to be more selective for the tetrodotoxin-resistant channel subtype Na(v)1.5/beta(1) (IC(50) = 447 +/- 32 nM). In addition to these results, molecular modeling indicates that subtle differences in toxin surfaces may relate to their different pharmacological profiles. Furthermore, an evolutionary trace analysis of these toxins and other structurally related three-disulfide spider toxins provides clues for the exploration of toxin-channel interaction and future structure-function research.