Ion channel auxiliary subunit: does one size fit all?

Ion channels can tailor their activity to the particular cellular context by incorporating auxiliary subunits that are channel-type specific. In this issue of Cell, Ávalos Prado et al. now find that a well-characterized voltage-gated K+ channel auxiliary subunit can also modulate the gating of Ca2+-activated Cl- channels.

Voltage Sensing Comes to Rest.

Voltage sensing by ion channels is the key event enabling the generation and propagation of electrical activity in excitable cells. In this issue of Cell, Wisedchaisri et al. provide a structural view of a voltage-gated sodium channel in its resting closed conformation.

Pore-modulating toxins exploit inherent slow inactivation to block K+ channels.

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

Reduced activity of GIRK1-containing heterotetramers is sufficient to affect neuronal functions, including synaptic plasticity and spatial learning and memory.

KEY POINTS: G-protein inwardly rectifying K+ (GIRK) channels consist of four homologous subunits (GIRK1-4) and are essential regulators of electrical excitability in the nervous system. GIRK2-null mice have been widely investigated for their distinct behaviour and altered depotentiation following long-term potentiation (LTP), whereas GIRK1 mice are less well characterized. Here we utilize a novel knockin mouse strain in which the GIRK1 subunit is fluorescently tagged with yellow fluorescent protein (YFP-GIRK1) and the GIRK1-null mouse line to investigate the role of GIRK1 in neuronal processes such as spatial learning and memory, locomotion and depotentiation following LTP. Neurons dissected from YFP-GIRK1 mice had significantly reduced potassium currents and this mouse line phenotypically resembled GIRK1-null mice, making it a 'functional knockdown' model of GIRK1-containing channels. YFP-GIRK1 and GIRK1-null mice had increased locomotion, reduced spatial learning and memory and blunted depotentiation following LTP. ABSTRACT: GIRK channels are essential for the slow inhibition of electrical activity in the nervous system and heart rate regulation via the parasympathetic system. The implications of individual GIRK isoforms in specific physiological activities are based primarily on studies conducted with GIRK-null mouse lines. Here we utilize a novel knockin mouse line in which YFP was fused in-frame to the N-terminus of GIRK1 (YFP-GIRK1) to correlate GIRK1 spatial distribution with physiological activities. These mice, however, displayed spontaneous seizure-like activity and thus were investigated for the origin of such activity. We show that GIRK tetramers containing YFP-GIRK1 are correctly assembled and trafficked to the plasma membrane, but are functionally impaired. A battery of behavioural assays conducted on YFP-GIRK1 and GIRK1-null (GIRK1-/- ) mice revealed similar phenotypes, including impaired nociception, reduced anxiety and hyperactivity in an unfamiliar environment. However, YFP-GIRK1 mice exhibited increased home-cage locomotion while GIRK1-/- mice did not. In addition, we show that the GIRK1 subunit is essential for intact spatial learning and memory and synaptic plasticity in hippocampal brain slices. This study expands our knowledge regarding the role of GIRK1 in neuronal processes and underlines the importance of GIRK1-containing heterotetramers.

One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Mapping the interaction site for a ß-scorpion toxin in the pore module of domain III of voltage-gated Na(+) channels.

Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. ß-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for ß-scorpion toxins on Na(V) channels. Binding of α- and ß-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.

Understanding the Molecular Regulation of Serotonin Receptor 5-HTR1B-ß-Arrestin1 Complex in Stress and Anxiety Disorders.

The serotonin receptor subtype 5-HTR1B is widely distributed in the brain with an important role in various behavioral implications including neurological conditions and psychiatric disorders. The neuromodulatory action of 5-HTR1B largely depends upon its arrestin mediated signaling pathway. In this study, we tried to investigate the role of unusually long intracellular loop 3 (ICL3) region of the serotonin receptor 5-HTR1B in interaction with ß-arrestin1 (Arr2) to compensate for the absence of the long cytoplasmic tail. Molecular modeling and docking tools were employed to obtain a suitable molecular conformation of the ICL3 region in complex with Arr2 which dictates the specific complex formation of 5-HTR1B with Arr2. This reveals the novel molecular mechanism of phosphorylated ICL3 mediated GPCR-arrestin interaction in the absence of the long cytoplasmic tail. The in-cell disulfide cross-linking experiments and molecular dynamics simulations of the complex further validate the model of 5-HTR1B-ICL3-Arr2 complex. Two serine residues (Ser281 and Ser295) within the 5-HTR1B-ICL3 region were found to be occupying the electropositive pocket of Arr2 in our model and might be crucial for phosphorylation and specific Arr2 binding. The alignment studies of these residues showed them to be conserved only across 5-HTR1B mammalian species. Thus, our studies were able to predict a molecular conformation of 5-HTR1B-Arr2 and identify the role of long ICL3 in the signaling process which might be crucial in designing targeted drugs (biased agonists) that promote GPCR-Arr2 signaling to deter the effects of stress and anxiety-like disorders.

Store-operated Ca2+ entry regulatory factor alters murine metabolic state in an age-dependent manner via hypothalamic pathways.

Store-operated calcium entry (SOCE) is a vital process aimed at refilling cellular internal Ca2+ stores and a primary cellular signaling driver for transcription factors' entry to the nucleus. SOCE-associated regulatory factor (SARAF)/TMEM66 is an endoplasmic reticulum (ER)-resident transmembrane protein that promotes SOCE inactivation and prevents Ca2+ overfilling of the cell. Here, we demonstrate that mice deficient in SARAF develop age-dependent sarcopenic obesity with decreased energy expenditure, lean mass, and locomotion without affecting food consumption. Moreover, SARAF ablation reduces hippocampal proliferation, modulates the activity of the hypothalamus-pituitary-adrenal (HPA) axis, and mediates changes in anxiety-related behaviors. Interestingly, selective SARAF ablation in the hypothalamus's paraventricular nucleus (PVN) neurons reduces old age-induced obesity and preserves locomotor activity, lean mass, and energy expenditure, suggesting a possible central control with a site-specific role for SARAF. At the cellular level, SARAF ablation in hepatocytes leads to elevated SOCE, elevated vasopressin-induced Ca2+ oscillations, and an increased mitochondrial spare respiratory capacity (SPC), thus providing insights into the cellular mechanisms that may affect the global phenotypes. These effects may be mediated via the liver X receptor (LXR) and IL-1 signaling metabolic regulators explicitly altered in SARAF ablated cells. In short, our work supports both central and peripheral roles of SARAF in regulating metabolic, behavioral, and cellular responses.

Elucidation of the molecular basis of selective recognition uncovers the interaction site for the core domain of scorpion alpha-toxins on sodium channels.

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.

Structure-function map of the receptor site for ß-scorpion toxins in domain II of voltage-gated sodium channels.

Voltage-gated sodium (Na(v)) channels are the molecular targets of ß-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of ß-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

Scorpion ß-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.

Production of recombinant venom peptides as tools for ion channel research.

Animal venom is a rich source for peptide toxins that bind and modulate the function of ion channels. Owing to their ability to bind receptor sites on the channel protein with high affinity and specificity, peptide neurotoxins have become an indispensable tool for ion channel research. Recent breakthroughs in structural biology and advances in computer simulations of biomolecules have sparked a new interest in animal toxins as probes of channel protein structure and function. Here, we focus on methods used to produce animal toxins for research purposes using recombinant expression. The specific challenges associated with heterologous production of venom peptides are discussed, and several methods targeting these issues are presented with an emphasis on E. coli based systems. An efficient protocol for the bacterial expression, folding, and purification of recombinant venom peptides is described.

Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation.

It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.

A Molecular Lid Mechanism of K+ Channel Blocker Action Revealed by a Cone Peptide.

Many venomous organisms carry in their arsenal short polypeptides that block K+ channels in a highly selective manner. These toxins may compete with the permeating ions directly via a "plug" mechanism or indirectly via a "pore-collapse" mechanism. An alternative "lid" mechanism was proposed but remained poorly defined. Here we study the Drosophila Shaker channel block by Conkunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and biophysical assays, implying discrete action modes. We show that while Conkunitzin-S1 binds tightly to the channel turret and acts via a "pore-collapse" mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a "molecular-lid". Our study provides an atomic description of the "lid" K+ blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.

Molecular requirements for recognition of brain voltage-gated sodium channels by scorpion alpha-toxins.

The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped ß-Sandwich Fold Important for SOCE Modulation.

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X-ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded ß-sandwich fold that includes a set of three conserved disulfide bonds, denoted the "SARAF-fold." The structure reveals a domain-swapped dimer in which the last two ß-strands (ß9 and ß10) are exchanged forming a region denoted the "SARAF luminal switch" that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation.

Non-sedating antihistamines block G-protein-gated inwardly rectifying K+ channels.

BACKGROUND AND PURPOSE: A second-generation antihistamine, terfenadine, is known to induce arrhythmia by blocking hERG channels. In this study, we have shown that terfenadine also inhibits the activity of G-protein-gated inwardly rectifying K+ (GIRK) channels, which regulate the excitability of neurons and cardiomyocytes. To clarify the underlying mechanism(s), we examined the effects of several antihistamines on GIRK channels and identified the structural determinant for the inhibition. EXPERIMENTAL APPROACH: Electrophysiological recordings were made in Xenopus oocytes and rat atrial myocytes to analyse the effects of antihistamines on various GIRK subunits (Kir 3.x). Mutagenesis analyses identified the residues critical for inhibition by terfenadine and the regulation of ion selectivity. The potential docking site of terfenadine was analysed by molecular docking. KEY RESULTS: GIRK channels containing Kir 3.1 subunits heterologously expressed in oocytes and native GIRK channels in atrial myocytes were inhibited by terfenadine and other non-sedating antihistamines. In Kir 3.1 subunits, mutation of Phe137, located in the centre of the pore helix, to the corresponding Ser in Kir 3.2 subunits reduced the inhibition by terfenadine. Introduction of an amino acid with a large side chain in Kir 3.2 subunits at Ser148 increased the inhibition. When this residue was mutated to a non-polar amino acid, the channel became permeable to Na+ . Phosphoinositide-mediated activity was also decreased by terfenadine. CONCLUSION AND IMPLICATIONS: The Phe137 residue in Kir 3.1 subunits is critical for inhibition by terfenadine. This study provides novel insights into the regulation of GIRK channels by the pore helix and information for drug design.

X-ray structure and mutagenesis of the scorpion depressant toxin LqhIT2 reveals key determinants crucial for activity and anti-insect selectivity.

Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.