Importance of position 8 in μ-conotoxin KIIIA for voltage-gated sodium channel selectivity

μ-Conotoxin KIIIA from Conus kinoshitai is a 16-residue peptide that acts as a potent pore blocker of several voltage-gated sodium channels (Na(v)). In order to obtain more selective blockers and to investigate the role of Trp at position 8, we substituted this residue with Arg, Gln and Glu. KIIIA and analogues were tested on a range of Na(v) expressed in Xenopus laevis oocytes. The rank order of potency for KIIIA was: rNa(v)1.4 ≥ rNa(v)1.2 > mNa(v)1.6 > rNa(v)1.3, with IC(50) values of 48 ± 6 nm, 61 ± 5 nm, 183 ± 31 nm and 3.6 ± 0.3 μm, respectively, whereas no effect was seen on hNa(v)1.5 and hNa(v)1.8 at a concentration of 10 μm. Replacement of Trp8 resulted in more selective blockers with a preference for neuronal sodium channels over the skeletal sodium channel. The activity on rNa(v)1.4 was reduced about 40-, 70- and 200-fold for [W8R]KIIIA, [W8Q]KIIIA and [W8E]KIIIA, respectively. All analogues showed a completely reversible block of rNa(v)1.2, as opposed to the partial reversibility of KIIIA. At saturating concentrations, complete block of rNa(v)1.2 was never achieved. The residual current was lower than 10%, except for [W8E]KIIIA. KIIIA had no effect on the voltage dependence of activation of rNa(v)1.2, whereas all analogues caused a depolarizing shift. Overall, this study shows that Trp8 is a key residue in the pharmacophore. Replacement of Trp8 enables more selective blockers to be obtained for neuronal sodium channels. Trp is a key determinant for the reversibility of block of rNa(v)1.2.     

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.     

Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.     

Design of Bioactive Peptides from Naturally Occurring μ-Conotoxin Structures

To date, cone snail toxins ("conotoxins") are of great interest in the pursuit of novel subtype-selective modulators of voltage-gated sodium channels (Na(v)s). Na(v)s participate in a wide range of electrophysiological processes. Consequently, their malfunctioning has been associated with numerous diseases. The development of subtype-selective modulators of Na(v)s remains highly important in the treatment of such disorders. In current research, a series of novel, synthetic, and bioactive compounds were designed based on two naturally occurring μ-conotoxins that target Na(v)s. The initial designed peptide contains solely 13 amino acids and was therefore named "Mini peptide." It was derived from the μ-conotoxins KIIIA and BuIIIC. Based on this Mini peptide, 10 analogues were subsequently developed, comprising 12-16 amino acids with two disulfide bridges. Following appropriate folding and mass verification, blocking effects on Na(v)s were investigated. The most promising compound established an IC(50) of 34.1 ± 0.01 nm (R2-Midi on Na(v)1.2). An NMR structure of one of our most promising compounds was determined. Surprisingly, this structure does not reveal an α-helix. We prove that it is possible to design small peptides based on known pharmacophores of μ-conotoxins without losing their potency and selectivity. These data can provide crucial material for further development of conotoxin-based therapeutics.     

Investigation of the relationship between the structure and function of Ts2, a neurotoxin from Tityus serrulatus venom

Scorpion toxins targeting voltage-gated sodium (Na(V)) channels are peptides that comprise 60-76 amino acid residues cross-linked by four disulfide bridges. These toxins can be divided in two groups (α and β toxins), according to their binding properties and mode of action. The scorpion α-toxin Ts2, previously described as a β-toxin, was purified from the venom of Tityus serrulatus, the most dangerous Brazilian scorpion. In this study, seven mammalian Na(V) channel isoforms (rNa(V)1.2, rNa(V)1.3, rNa(V)1.4, hNa(V)1.5, mNa(V)1.6, rNa(V)1.7 and rNa(V)1.8) and one insect Na(V) channel isoform (DmNa(V)1) were used to investigate the subtype specificity and selectivity of Ts2. The electrophysiology assays showed that Ts2 inhibits rapid inactivation of Na(V)1.2, Na(V)1.3, Na(V)1.5, Na(V)1.6 and Na(V)1.7, but does not affect Na(V)1.4, Na(V)1.8 or DmNa(V)1. Interestingly, Ts2 significantly shifts the voltage dependence of activation of Na(V)1.3 channels. The 3D structure of this toxin was modeled based on the high sequence identity (72%) shared with Ts1, another T. serrulatus toxin. The overall fold of the Ts2 model consists of three β-strands and one α-helix, and is arranged in a triangular shape forming a cysteine-stabilized α-helix/β-sheet (CSαβ) motif.     

Neuronally micro-conotoxins from Conus striatus utilize an alpha-helical motif to target mammalian sodium channels

Mu-conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of mu-conotoxins SIIIA and SIIIB by (125)I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced (125)I-TIIIA from native rat brain Na(v)1.2 (IC(50) values 10 and 5 nm, respectively) and muscle Na(v)1.4 (IC(50) values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na(v)1.2 and Na(v)1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC(50) 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na(v)1.2 versus 1.4 selectivity (136-fold). (1)H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an alpha-helical motif across residues 11-16 that is not found in larger mu-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller mu-conotoxin pharmacophore.     

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA,PIIIA, and KIIIA in a NavAb-based model of Nav1.4

Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Noradrenaline Antagonizes and Ouabain Potentiates the Effects of iV-Methyl-D-Aspartate on Rat Cerebellar Cyclic GMP Production

Noradrenaline potently antagonizes the effects of N-methyl-D-aspartate (NMDA) (80 microM) on cyclic GMP production in immature rat cerebellar slices in vitro (IC50 = 0.6 microM). The effect is stereospecific (D-noradrenaline, IC50 = 100 microM), and also observed with adrenaline (IC50 = 0.5 microM) and isoprenaline (IC50 = 1.2 microM). The alpha 1-adrenoceptor agonists methoxamine or phenylephrine or the mixed alpha 1/alpha 2 agonists oxymetazoline or xylometazoline (100 microM) do not block the effects of NMDA, but the alpha 2-adrenoceptor agonist clonidine is weakly active (IC50 = 200 microM). Salbutamol and terbutaline were also inactive except at high concentrations (300 microM), as were a number of other catechol and phenylethylamine derivatives. The antagonistic effects of noradrenaline on the NMDA response were insensitive to phentolamine, atenolol, or propranolol (up to 100 microM), but were blocked by the alpha 2 antagonist idazoxan (1-10 microM). The Na+,K+-ATPase inhibitor ouabain (0.1-10 microM) markedly potentiates the effects of NMDA in this model, and also antagonizes and reverses the ability of noradrenaline (10 microM) to block the effects of NMDA. The results suggest that noradrenaline and Na+,K+-ATPase activity have potent modulatory effects on the NMDA response.     

A disubstituted succinamide is a potent sodium channel blocker with efficacy in a rat pain model

Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In a high-throughput functional assay, a novel sodium channel (Na(V)) blocker, N-[[2'-(aminosulfonyl)biphenyl-4-yl]methyl]-N'-(2,2'-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of Na(V)1.2, Na(V)1.5, and Na(V)1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of Na(V)1.2 was 1.2 and 0.14 microM, respectively. BPBTS blocked Na(V)1.7 and Na(V)1.2 with similar potency, whereas block of Na(V)1.5 was slightly more potent. The slow tetrodotoxin-resistant Na(+) current in small-diameter DRG neurons was also potently blocked by BPBTS. [(3)H]BPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K(d) = 6.1 nM), and in membranes derived from HEK cells stably expressing Na(V)1.5 (K(d) = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.     

N- and C-terminal extensions of μ-conotoxins increase potency and selectivity for neuronal sodium channels

μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity.     

The Janus-faced atracotoxins are specific blockers of invertebrate K(Ca) channels

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.     

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.     

Morphine decreases the voltage sensitivity of the slow sodium channels]

Morphine was shown to decrease in a dose-dependent manner the effective charge transfer in tetrodotoxin-resistant (TTXr) sodium channel activation system in short-term cultured dorsal root ganglion cells. Morphine seems to interact with opioid receptors because of total block of the binding by naloxone and naltrexone. Neither activating, nor inhibiting G-protein agents exerted any effect on this process. The morphine signal was blocked by extracellular application of 2 x 10(-4) M ouabain. The findings suggest existence of sodium signalling pathway involving receptors, Na+, K(+)-ATPase and the TTXr sodium channels.     

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na(+) channels with higher affinities. However, an alternative view suggests that activation of Na(+) channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na(+) channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC(50)) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 +/- 3.3 microM (n = 5) and 81.7 +/- 10.6 microM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC(50) values for resting-channel block at -140 mV were >12-fold higher than those for open-channel block. With 300 microM benzocaine, rapid time-dependent block (tau approximately 0.8 ms) of inactivation-deficient Na(+) currents occurred at +30 mV, but such a rapid time-dependent block was not evident at -30 mV. The peak current at -30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na(+) channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.     

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.     

The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels

Na(+)/Ca(2+) exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca(2+) homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca(2+) influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC(50)=0.46 microM), TRPC6 (IC(50)=0.71 microM), and TRPC5 (IC(50)=1.38 microM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry was nearly completely suppressed by 10 microM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.     

Calmodulin regulation of Nav1.4 current: role of binding to the carboxyl terminus

Calmodulin (CaM) regulates steady-state inactivation of sodium currents (Na(V)1.4) in skeletal muscle. Defects in Na current inactivation are associated with pathological muscle conditions such as myotonia and paralysis. The mechanisms of CaM modulation of expression and function of the Na channel are incompletely understood. A physical association between CaM and the intact C terminus of Na(V)1.4 has not previously been demonstrated. FRET reveals channel conformation-independent association of CaM with the C terminus of Na(V)1.4 (CT-Na(V)1.4) in mammalian cells. Mutation of the Na(V)1.4 CaM-binding IQ motif (Na(V)1.4(IQ/AA)) reduces cell surface expression of Na(V)1.4 channels and eliminates CaM modulation of gating. Truncations of the CT that include the IQ region abolish Na current. Na(V)1.4 channels with one CaM fused to the CT by variable length glycine linkers exhibit CaM modulation of gating only with linker lengths that allowed CaM to reach IQ region. Thus one CaM is sufficient to modulate Na current, and CaM acts as an ancillary subunit of Na(V)1.4 channels that binds to the CT in a conformation-independent fashion, modulating the voltage dependence of inactivation and facilitating trafficking to the surface membrane.     

The cross channel activities of spider neurotoxin huwentoxin-I on rat dorsal root ganglion neurons

In this paper, we investigated the action of huwentoxin-I (HWTX-I) purified from the venom of the Chinese bird spider Ornithoctonus huwena on Ca(2+), Na(+) channels of adult rat dorsal root ganglion (DRG) neurons. The results showed that huwentoxin-I could reduce the peak currents of N-type Ca(2+) channels (IC(50) approximately 100 nM) and TTX-S Na(+) channels (IC(50) approximately 55 nM), whereas no effect was detected on TTX-R Na(+) channels. The comparative studies indicated that the selectivity of HWTX-I on Ca(2+) channels was higher that of MVIIA and approximately the same as that of GVIA. HWTX-I is the first discovered toxin with the cross channel activities from the spider O. huwena venom similar to micro O-conotoxins MrVIA and MrVIB.