An integrative approach to the facile functional classification of dorsal root ganglion neuronal subclasses.

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] i Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the Kv1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional Kv1.1/Kv1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population.

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Subtype-specific block of voltage-gated K+ channels by µ-conopeptides.

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Biochemical characterization of kappaM-RIIIJ, a Kv1.2 channel blocker: evaluation of cardioprotective effects of kappaM-conotoxins.

Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC(50) = 33 nm) to Kv1.2 than kappaM-RIIIK (IC(50) = 352 nm). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK, whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC(50) = 680 nm) than that of kappaM-RIIIJ (IC(50) = 3.15 mum). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

In vitro developed spontaneously contracting cardiomyocytes from rainbow trout as a model system for human heart research.

BACKGROUND/AIMS: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. METHODS: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra - and extracellular recordings. RESULTS: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. CONCLUSION: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

Molecular and Functional Differences between Heart mKv1.7 Channel Isoforms.

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

Discovery and characterization of the short kappaA-conotoxins: a novel subfamily of excitatory conotoxins.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3.

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Molecular interaction of delta-conotoxins with voltage-gated sodium channels.

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore.

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.

Fibroblast Growth Factor Enhances Long-term Potentiation in the Hippocampal Slice.

Recently we reported that perfusion of hippocampal slices with epidermal growth factor (EGF) lead to enhancement of potentiated responses after tetanic stimulation. In the present study we report that basic fibroblast growth factor (FGF) can also lead to an enhancement of potentiated responses. FGF is a mitogen for several cell types and exhibits neurotrophic effects on neurons of the central nervous system (CNS). Rat hippocampal slices were perfused with FGF at a concentration of 10-9 M. During extra- and intracellular recordings in the CA1-region, the addition of FGF to the perfusing medium produced no change in evoked responses if single pulse or paired pulse stimulation was used. Furthermore FGF had no influence on the resting membrane potential and input resistance. However, after tetanic stimulation, FGF-treated slices showed an increase in the magnitude of potentiation compared to control slices. Taken together with the EGF data these results support the hypothesis that growth factors like FGF with neurotrophic potential on CNS-neurons can influence synaptic efficacy. Furthermore these results show that factors which are able to modulate developmental plasticity and regenerative plasticity can also modulate synaptic plasticity.

Sodium channel toxins--receptor targeting and therapeutic potential.

Sodium channels underlie propagated electrical signalling in most excitable cells, including neurons and the myocytes of skeletal muscle and heart. These proteins are targeted by a variety of current therapeutic drugs to combat such maladies as pain, myotonias, epilepsies and cardiac arrhythmias. Typically, these problems are associated with overactivity of sodium channels leading to hyperexcitability in the relevant tissue. More than ten distinct but closely related molecular isoforms of mammalian sodium channel are now known to be specifically expressed in different cell types and tissues. Therapeutic attenuation of sodium channel activity must be effected with great precision in both targeting and the degree of reduction in channel activity if a malfunction is to be corrected without introducing deleterious or even catastrophic side effects. Numerous natural toxins have evolved to target sodium channels, either by blocking current through the pore or by modifying channel gating. Among the well studied toxins, the peptide conotoxins from cone snail venoms show a remarkable ability to discriminate among closely related forms of sodium channel, as well as exhibiting a variety of modes of action. Here, we examine the molecular basis of action of different Na channel targeted conotoxins and explore their potential as models for the future design of more specifically targeted drugs.

Identification of a mammalian target of kappaM-conotoxin RIIIK.

Despite the great variability of the conus peptides characterized until now only relatively few have been identified that interact with K+ channels. kappaM-conotoxin RIIIK (kappaM-RIIIK) is a 24 amino acid peptide from Conus radiatus, which is structurally similar to micro-conotoxin GIIIA, a peptide known to block specifically skeletal muscle Na+ channels. Recently, it has been shown that kappaM-RIIIK does not interact with Na) channels, but inhibits Shaker potassium channels expressed in Xenopus oocytes. It was demonstrated that kappaM-RIIIK binds to the pore region of Shaker channels and a teleost homologue of the Shaker channel TSha1 was identified as a high affinity target of the toxin. In contrast the mammalian Shaker-homologues Kv1.1, Kv1.3, Kv1.4 are not affected by the toxin. In this study the activity of kappaM-RIIIK on other mammalian Kv1 K+ channels expressed in Xenopus oocytes was investigated. We demonstrate that kappaM-conotoxin RIIIK up to 5 microM exhibits no significant effect on Kv1.5 and Kv1.6 mediated currents, but the human Kv1.2 K+ channel is blocked by this peptide. The binding of kappaM-RIIIK to Kv1.2 channels is state dependent with an IC50 for the closed state of about 200 nM and for the open state of about 400 nM at a test potential of 0 mV. kappaM-conotoxin RIIIK is the first conotoxin described to block human Kv1.2 potassium channels.

Pharmacology of voltage-gated potassium channel Kv1.5--impact on cardiac excitability.

Voltage activated potassium (Kv) channels are intensely investigated targets within the pharmacological strategies to treat cardiac arrhythmia. For atrial fibrillation (AF) substances inhibiting the ultra rapid outward rectifying Kv current (IKur) and its underlying Kv1.5 channel have been developed. Here we describe potential limitations of this approach with respect to critical parameters of Kv channel pharmacology. In healthy tissue IKur/Kv1.5 inhibition can unexpectedly lead to action potential shortening with corresponding arrhythmogenic effects. In tissue with chronic AF, electrical remodeling occurs which is accompanied with changes in ion channel expression and composition. As a consequence atrial tissue exhibits a different pharmacological fingerprint. New strategies to obtain more mechanistic insight into drug target interaction are needed for better understanding the pharmacological potential of IKur/Kv1.5 inhibition for AF treatment.

A family of excitatory peptide toxins from venomous crassispirine snails: using Constellation Pharmacology to assess bioactivity.

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types.

Organotypic slice culture from human adult ventricular myocardium.

AIMS: Cardiovascular research requires complex and functionally intact experimental models. Due to major differences in the cellular and subcellular composition of the myocardium between species, the use of human heart tissue is highly desirable. To enhance the experimental use of the human myocardium, we established methods for the preparation of vital tissue slices from the adult ventricular myocardium as well as conditions for their long-term preservation in organotypic culture. METHODS AND RESULTS: Human ventricular heart samples were derived from surgical specimens excised during a therapeutic Morrow myectomy and cut into 300 µm thick slices. Slices were either characterized in acute experiments or cultured at a liquid-air interface. Viability and functionality were proven by viability staining, enzyme activity tests, intracellular potential recordings, and force measurements. Precision-cut slices showed high viability throughout 28 days in culture and displayed typical cardiomyocyte action potential characteristics, which enabled pharmacological safety testing on the rapid component of the delayed rectifier potassium current (I(Kr)) and ATP-dependent potassium channels throughout the whole culture period. Constant expression of major ion channels was confirmed by quantitative PCR. Acute slices developed excitation-dependent contractions with a clear preload dependency and a ß-adrenergic response. Contractility and myosin light chain expression decreased during the first days in culture but reached a steady state with reactivity upon ß-adrenergic stimulation being preserved. CONCLUSION: Organotypic heart slices represent a multicellular model of the human myocardium and a novel platform for studies ranging from the investigation of molecular interactions to tissue engineering.

Block of Kv1.7 potassium currents increases glucose-stimulated insulin secretion.

Glucose-stimulated insulin secretion (GSIS) relies on repetitive, electrical spiking activity of the beta cell membrane. Cyclic activation of voltage-gated potassium channels (K(v) ) generates an outward, 'delayed rectifier' potassium current, which drives the repolarizing phase of each spike and modulates insulin release. Although several K(v) channels are expressed in pancreatic islets, their individual contributions to GSIS remain incompletely understood. We take advantage of a naturally occurring cone-snail peptide toxin, Conkunitzin-S1 (Conk-S1), which selectively blocks K(v) 1.7 channels to provide an intrinsically limited, finely graded control of total beta cell delayed rectifier current and hence of GSIS. Conk-S1 increases GSIS in isolated rat islets, likely by reducing K(v) 1.7-mediated delayed rectifier currents in beta cells, which yields increases in action potential firing and cytoplasmic free calcium. In rats, Conk-S1 increases glucose-dependent insulin secretion without decreasing basal glucose. Thus, we conclude that K(v) 1.7 contributes to the membrane-repolarizing current of beta cells during GSIS and that block of this specific component of beta cell K(v) current offers a potential strategy for enhancing GSIS with minimal risk of hypoglycaemia during metabolic disorders such as Type 2 diabetes.

Toxins from cone snails: properties, applications and biotechnological production.

Cone snails are marine predators that use venoms to immobilize their prey. The venoms of these mollusks contain a cocktail of peptides that mainly target different voltage- and ligand-gated ion channels. Typically, conopeptides consist of ten to 30 amino acids but conopeptides with more than 60 amino acids have also been described. Due to their extraordinary pharmacological properties, conopeptides gained increasing interest in recent years. There are several conopeptides used in clinical trials and one peptide has received approval for the treatment of pain. Accordingly, there is an increasing need for the production of these peptides. So far, most individual conopeptides are synthesized using solid phase peptide synthesis. Here, we describe that at least some of these peptides can be obtained using prokaryotic or eukaryotic expression systems. This opens the possibility for biotechnological production of also larger amounts of long chain conopeptides for the use of these peptides in research and medical applications.

Tyrosine-rich conopeptides affect voltage-gated K+ channels.

Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.

muO conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2.

The muO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na(V)1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which muO-conotoxins affect Na(V) channels. Rat Na(V)1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily mu-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na(V) channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion beta-toxins. In fact, MrVIA functionally competed with the scorpion beta-toxin Ts1 from Tityus serrulatus, while it did not show competition with mu-GIIIA. Ts1 and mu-GIIIA did not compete either. Thus, similar to scorpion beta-toxins, muO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na(V) channels. They "block" Na(+) flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.