Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: 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.     

Molecular basis of isoform-specific micro-conotoxin block of cardiac,skeletal muscle,and brain Na+ channels

mu-Conotoxins (mu-CTXs) block skeletal muscle Na(+) channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na(+) channels. Although a number of conserved pore residues are recognized as critical determinants of mu-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) Na(+) channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.     

Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.     

Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats

We recently reported that reactive oxygen species (ROS) plays an excitatory role in modulation of the exercise pressor reflex (EPR) in normal rats. In this study, we further tested two independent hypotheses: 1) ROS interacts with EPR-related ionotropic receptors such as the purinergic receptors (P(2)) and transient receptor potential vanilloid 1 receptors (TRPV1) to indirectly modulate the EPR function; 2) ROS directly affects excitability of muscle afferents by modulating the voltage-gated sodium (Na(v)) channels. To test the first hypothesis, we performed animal experiments to investigate the effect of the SOD mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) on the pressor response to hindlimb intra-arterial (IA) injection of either α,β-methylene ATP (a P(2X) agonist) or capsaicin (a TRPV1 agonist) in decerebrate rats. To test the second hypothesis, we used the patch-clamp technique to determine the effect of ROS on Na(v) channels on the soma of muscle afferents. We also performed local microinjection of a sodium channel blocker, tetrodotoxin (TTX), into ipsilateral L4/L5 dorsal root ganglia (DRGs) to investigate whether the blockade of Na(v) channels by TTX affects the EPR function. We found that Tempol did not affect the pressor response to injection of either capsaicin or α,β-methylene ATP but significantly decreased the Na(v) current in small and medium-sized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons. A membrane-permeant superoxide dismutase, polyethylene glycol (PEG)-SOD, had an effect on the Na(v) current in these neurons similar to that of Tempol. Microinjection of TTX into L4/L5 DRGs dramatically attenuated the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots. These data suggest that ROS modulates the EPR by affecting the activity of the Na(v) channels on muscle afferents.     

Chinese-scorpion (Buthus martensi Karsch) toxin BmK alphaIV, a novel modulator of sodium channels: from genomic organization to functional analysis

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.     

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+) (Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v) contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na(v) channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na(v) channels in vivo.     

Na v1.4 and Na v1.5 are modulated differently during muscle immobilization and contractile phenotype conversion

Muscle immobilization leads to modification in its fast/slow contractile phenotype. Since the properties of voltage-gated sodium channels (Na(v)) are different between "fast" and "slow" muscles, we studied the effects of immobilization on the contractile properties and the Na(v) of rat peroneus longus (PL). The distal tendon of PL was cut and fixed to the adjacent bone at neutral muscle length. After 4 or 8 wk of immobilization, the contractile and the Na(v) properties were studied and compared with muscles from control animals (Student's t-test). After 4 wk of immobilization, PL showed a faster phenotype with a rightward shift of the force-frequency curve and a decrease in both the Burke's index of fatigability and the tetanus-to-twitch ratio. These parameters showed opposite changes between 4 and 8 wk of immobilization. The maximal sodium current in 4-wk immobilized fibers was higher compared with that of control fibers (11.5 ± 1.2 vs. 7.8 ± 0.8 nA, P = 0.008), with partial recovery to the control values in 8-wk immobilized fibers (8.6 ± 0.7 nA, P = 0.48). In the presence of tetrodotoxin, the maximal residual sodium current decreased continuously throughout immobilization. Using the Western blot analysis, Na(v)1.4 expression showed a transient increase in 4-wk muscle, whereas Na(v)1.5 expression decreased during immobilization. Our results indicate that a muscle immobilized at optimal functional length with the preservation of neural inputs exhibits a transient fast phenotype conversion. Na(v)1.4 expression and current are related to the contractile phenotype variation.     

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.     

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.     

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.     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells

We have investigated the effect of tunicamycin (TM), an inhibitor of protein glycosylation, on surface Na+ channels in cultured chick skeletal muscle cells. The expression of Na+ channels, estimated by the measurement of batrachotoxin (BTX)-activated 22Na+ uptake, was found to be significantly diminished after exposure of muscle cells to TM. This effect is partially reversed by the protease inhibitor leupeptin and is associated with a markedly enhanced rate of disappearance of Na+ channels from the surface of TM-treated cells. Our findings suggest that protein glycosylation contributes to the metabolic stability of voltage-sensitive Na+ channels.     

Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region

In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.     

A mutation in telethonin alters Nav1.5 function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.     

Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets

Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575-585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8-5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 microg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 microg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 microg/ml) was similar to that obtained with 16.5 microg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in beta-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na(+) channels located in the beta-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na(+) channels in the mechanism of insulin secretion.     

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.     

Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.