Voltage-gated Sodium Channel Isoforms Research Articles

Vector-mediated release of GABA attenuates pain-related behaviors and reduces Na V1.7 in DRG neurons

Pain is a common and debilitating accompaniment of neuropathy that occurs as a complication of diabetes. In the current study, we examined the effect of continuous release of gamma amino butyric acid (GABA), achieved by gene transfer of glutamic acid decarboxylase (GAD67) to dorsal root ganglia (DRG) in vivo using a non-replicating herpes simplex virus (HSV)-based vector (vG) in a rat model of painful diabetic neuropathy (PDN). Subcutaneous inoculation of vG reduced mechanical hyperalgesia, thermal hyperalgesia and cold allodynia in rats with PDN. Continuous release of GABA from vector transduced cells in vivo prevented the increase in the voltage-gated sodium channel isoform 1.7 (Na V1.7) protein that is characteristic of PDN. In vitro, infection of primary DRG neurons with vG prevented the increase in Na V1.7 resulting from exposure to hyperglycemia. The effect of vector-mediated GABA on Na V1.7 levels in vitro was blocked by phaclofen but not by bicuculline, a GABA B receptor effect that was blocked by pertussis toxin-(PTX) interference with Gα( i/o) function. Taken in conjunction with our previous observation that continuous activation of delta opioid receptors by vector-mediated release of enkephalin also prevents the increase in Na V1.7 in DRG exposed to hyperglycemia in vitro or in vivo, the observations in this report suggest a novel common mechanism through which activation of G protein coupled receptors (GPCR) in DRG neurons regulate the phenotype of the primary afferent.

Quantitative RT-PCR analyses of five evolutionary conserved genes in Alligator brains during development

Gene expression was investigated in the major brain subdivisions (telencephalon, diencephalon, midbrain and hindbrain) in a representative reptile, Alligator mississipiensis, during the later stages of embryonic development. The following genes were examined: voltage-gated sodium channel isoforms: NaV1.1 and NaV1.2; synaptic vesicle 2a (SV2a); synaptophysin; and calbindin 2. With the exception of synaptophysin, which was only expressed in the telencephalon, all genes were expressed in all brain regions sampled at the time periods examined. For NaV1.1, gene expression varied according to brain area sampled. When compared with NaV1.1, the pattern of NaV1.2 gene expression differed appreciably. The gene expression of SV2a was the most robust of any of the genes examined. Of the other genes examined, although differences were noted, no statistically significant changes were found either between brain part or time interval. Although limited, the present analysis is the first quantitative mRNA gene expression study in any reptile during development. Together with future experiments of a similar nature, the present gene expression results should determine which genes are expressed in major brain areas at which times during development in Alligator. When compared with other amniotes, these results will prove useful for determining how gene expression during development influences adult brain structure.

Nav1.8 channelopathy in mutant mice deficient for myelin protein zero is detrimental to motor axons

Myelin protein zero mutations were found to produce Charcot-Marie-Tooth disease phenotypes with various degrees of myelin impairment and axonal loss, ranging from the mild 'demyelinating' adult form to severe and early onset forms. Protein zero deficient homozygous mice ( ) show a severe and progressive dysmyelinating neuropathy from birth with compromised myelin compaction, hypomyelination and distal axonal degeneration. A previous study using immunofluorescence showed that motor nerves deficient of myelin protein zero upregulate the Na(V)1.8 voltage gated sodium channel isoform, which is normally present only in restricted populations of sensory axons. The aim of this study was to investigate the function of motor axons in protein zero-deficient mice with particular emphasis on ectopic Na(V)1.8 voltage gated sodium channel. We combined 'threshold tracking' excitability studies with conventional nerve conduction studies, behavioural studies using rotor-rod measurements, and histological measures to assess membrane dysfunction and its progression in protein zero deficient homozygous mutants as compared with age-matched wild-type controls. The involvement of Na(V)1.8 was investigated by pharmacologic block using the subtype-selective Na(V)1.8 blocker A-803467 and chronically in Na(V)1.8 knock-outs. We found that in the context of dysmyelination, abnormal potassium ion currents and membrane depolarization, the ectopic Na(V)1.8 channels further impair the motor axon excitability in protein zero deficient homozygous mutants to an extent that precipitates conduction failure in severely affected axons. Our data suggest that a Na(V)1.8 channelopathy contributed to the poor motor function of protein zero deficient homozygous mutants, and that the conduction failure was associated with partially reversible reduction of the electrically evoked muscle response and of the clinical function as indicated by the partial recovery of function at rotor-rod measurements. As a consequence of these findings of partially reversible dysfunction, we propose that the Na(V)1.8 voltage gated sodium channel should be considered as a novel therapeutic target for Charcot-Marie-Tooth disease.

Regulation of the Spontaneous Augmentation of NaV1.9 in Mouse Dorsal Root Ganglion Neurons: Effect of PKA and PKC Pathways

Sensory neurons in the dorsal root ganglion express two kinds of tetrodotoxin resistant (TTX-R) isoforms of voltage-gated sodium channels, NaV1.8 and NaV1.9. These isoforms play key roles in the pathophysiology of chronic pain. Of special interest is NaV1.9: our previous studies revealed a unique property of the NaV1.9 current, i.e., the NaV1.9 current shows a gradual and notable up-regulation of the peak amplitude during recording (“spontaneous augmentation of NaV1.9”). However, the mechanism underlying the spontaneous augmentation of NaV1.9 is still unclear. In this study, we examined the effects of protein kinases A and C (PKA and PKC), on the spontaneous augmentation of NaV1.9. The spontaneous augmentation of the NaV1.9 current was significantly suppressed by activation of PKA, whereas activation of PKA did not affect the voltage dependence of inactivation for the NaV1.9 current. On the contrary, the finding that activation of PKC can affect the voltage dependence of inactivation for NaV1.9 in the perforated patch recordings, where the augmentation does not occur, suggests that the effects of PMA are independent of the augmentation process. These results indicate that the spontaneous augmentation of NaV1.9 was regulated directly by PKA, and indirectly by PKC.

Voltage‐gated sodium channel isoform‐specific effects of pompilidotoxins

Pompilidotoxins (PMTXs, alpha and beta) are small peptides consisting of 13 amino acids purified from the venom of the solitary wasps Anoplius samariensis (alpha-PMTX) and Batozonellus maculifrons (beta-PMTX). They are known to facilitate synaptic transmission in the lobster neuromuscular junction, and to slow sodium channel inactivation. By using beta-PMTX, alpha-PMTX and four synthetic analogs with amino acid changes, we conducted a thorough study of the effects of PMTXs on sodium current inactivation in seven mammalian voltage-gated sodium channel (VGSC) isoforms and one insect VGSC (DmNa(v)1). By evaluating three components of which the inactivating current is composed (fast, slow and steady-state components), we could distinguish three distinct groups of PMTX effects. The first group concerned the insect and Na(v)1.6 channels, which showed a large increase in the steady-state current component without any increase in the slow component. Moreover, the dose-dependent increase in this steady-state component was correlated with the dose-dependent decrease in the fast component. A second group of effects concerned the Na(v)1.1, Na(v)1.2, Na(v)1.3 and Na(v)1.7 isoforms, which responded with a large increase in the slow component, and showed only a small steady-state component. As with the first group of effects, the slow component was dose-dependent and correlated with the decrease in the fast component. Finally, a third group of effects concerned Na(v)1.4 and Na(v)1.5, which did not show any change in the slow or steady-state component. These data shed light on the complex and intriguing behavior of VGSCs in response to PMTXs, helping us to better understand the molecular determinants explaining isoform-specific effects.

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

The Na(v)1.2 and Na(v)1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary beta1 subunit accelerated inactivation of both the Na(v)1.2 and Na(v)1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the alpha subunit. By constructing chimeric channels between Na(v)1.2 and Na(v)1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Na(v)1.2 carboxyl terminus caused faster inactivation in the Na(v)1.3 backbone, and the Na(v)1.3 carboxyl terminus caused slower inactivation in the Na(v)1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Na(v)1.2 and the Na(v)1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Na(v)1.3 lysine 1826 in this region to its Na(v)1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Na(v)1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Na(v)1.2. The reverse Na(v)1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Na(v)1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Na(v)1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.

Sodium-Calcium Exchanger and Multiple Sodium Channel Isoforms in Intra-Epidermal Nerve Terminals

BackgroundNociception requires transduction and impulse electrogenesis in nerve fibers which innervate the body surface, including the skin. However, the molecular substrates for transduction and action potential initiation in nociceptors are incompletely understood. In this study, we examined the expression and distribution of Na+/Ca2+ exchanger (NCX) and voltage-gated sodium channel isoforms in intra-epidermal free nerve terminals.ResultsSmall diameter DRG neurons exhibited robust NCX2, but not NCX1 or NCX3 immunolabeling, and virtually all PGP 9.5-positive intra-epidermal free nerve terminals displayed NCX2 immunoreactivity. Sodium channel NaV1.1 was not detectable in free nerve endings. In contrast, the majority of nerve terminals displayed detectable levels of expression of NaV1.6, NaV1.7, NaV1.8 and NaV1.9. Sodium channel immunoreactivity in the free nerve endings extended from the dermal boundary to the terminal tip. A similar pattern of NCX and sodium channel immunolabeling was observed in DRG neurons in vitro.ConclusionsNCX2, as well as NaV1.6, NaV1.7, NaV1.8 and NaV1.9, are present in most intra-epidermal free nerve endings. The presence of NCX2, together with multiple sodium channel isoforms, in free nerve endings may have important functional implications.

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na + channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na + channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na + channels (Na v1. x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na v1. x channels and all are 46–49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na + channel protein. This region is indeed responsible for the normal Na + currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.

Recent Advances in the Medicinal Chemistry of Sodium Channel Blockers and their Therapeutic Potential

The voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability. Abnormal activity of sodium channels is related to several pathological processes, including cardiac arrhythmias, epilepsy, chronic pain, neurodegenerative diseases and spasticity. In view of this, VGSCs are considered important therapeutic targets for the treatment of these disorders. To date, nine VGSC isoforms have been identified and have a distinct pattern of expression within the human body. In addition, VGSCs also have distinct electrophysiological profiles with differing activation and inactivation states. As such, there is a concerted effort to develop not only isoform selective antagonists, but also antagonists that exhibit state selectivity, particularly to the inactivated state of the channel. This review will provide a brief historical prospective and will primarily focus on recent advances in the development of isoform specific and state selective sodium channel antagonists and the medicinal chemistry involved, surveying the emerging therapeutic fields.

Biophysical Characterization Of Duloxetine Activity On Voltage-gated Sodium Channels Involved In Pain Transmission

Duloxetine, an inhibitor of the serotonin-norepinephrine reuptake system, is widely used for the treatment of major depression and has also been found to be effective in reducing neuropathic pain. This latter effect is believed to be related to the role of the two neurotransmitters in inhibiting pain signals at the level of spinal and supraspinal neural circuits. However the possibility that duloxetine also acts through alternative mechanisms has not yet been investigated. In this study we focused on the potential effect of duloxetine on neuronal isoforms of voltage-gated sodium channels (NaV1.3 and NaV1.7) that have a pivotal role in the generation and propagation of pain signals. Conventional whole-cell patch-clamp recordings were performed on CHO-cells expressing hNaV1.3 and hNaV1.7. Duloxetine exerted a concentration-dependent resting block, measured by the reduction of the sodium current elicited by a single depolarizing step from -100mV to 0mV for 10 ms, with IC50 values of 5 and 16 microM for NaV1.3 and NaV1.7, respectively. These values were halved at holding potentials that reduce channel availability of both channels by 50%, indicating a preference for the inactivated state of sodium channels. Furthermore we observed that 10 microM duloxetine induced a 10mV negative shift of the steady state inactivation curves, and preliminary results indicate that this effect is correlated with stabilization of slow inactivation. Finally, for both sodium channel isoforms, an additional 20% inhibition over the resting block was observed at concentrations close to the IC50 when the stimulation frequency was increased up to 10Hz. Thus, the antidepressant duloxetine exhibits most of the state-dependent mechanisms of a classic sodium channel blocker. These results may add valuable information about the pharmacological spectrum of duloxetine, an issue of high clinical impact considering its increasingly widespread use.

ProTxII Interacts Specifically with the Domain II Voltage Sensor of NaV1.4 Modifying Gating Without Immobilization

ProTxII, a peptide extracted from the venom of the tarantula Thrixopelma pruriens, binds to multiple voltage-gated sodium channel isoforms. In NaV1.2 ProTxII reduces the sodium conductance and decreases total gating charge (Sokolov et al., Mol. Pharm. 73:1020). In this isoform the toxin's effect could be partially reversed with strong depolarization indicating interactions with one or more of the voltage sensors. Mutagenesis data further implicated the domain II voltage sensor in the toxin-channel interaction in NaV1.2.

Role of the amino and carboxy termini in isoform‐specific sodium channel variation

Na(v)1.2 and Na(v)1.6 are two voltage-gated sodium channel isoforms found in adult CNS neurons. These isoforms differ in their electrophysiological properties, even though the major regions that are known to be involved in channel activation and inactivation are conserved between them. To determine if the terminal domains of these channels contributed to their activation and fast inactivation differences, we constructed chimeras between the two isoforms and characterized their electrophysiological properties. Exchanging the N-terminal 205 amino acids of Na(v)1.6 and the corresponding 202 amino acids of Na(v)1.2 completely swapped the V_(1)/(2) of steady-state activation between the Na(v)1.2 and Na(v)1.6 channels in an isoform-specific manner. Exchanging the C-terminal 436 amino acids of Na(v)1.6 and the corresponding region of Na(v)1.2 altered the voltage dependence and kinetics of steady-state inactivation, but the changes did not reflect a direct transfer of inactivation properties between the two isoforms. Finally, the N- and C-terminal domains from Na(v)1.6 demonstrated functional cooperation. These results suggest that the terminal sequences of the sodium channel are important for isoform-specific differences between the channels.

Continuous -Opioid Receptor Activation Reduces Neuronal Voltage-Gated Sodium Channel (NaV1.7) Levels through Activation of Protein Kinase C in Painful Diabetic Neuropathy

The Na(V)1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased Na(V)1.7 in dorsal root ganglia (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus-based vector expressing proenkephalin reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG Na(V)1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mm glucose for 18 h also demonstrated an increase in Na(V)1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on Na(V)1.7 production in vitro was mimicked by exposure to PMA and blocked by the myristolated PKC inhibitor 20-28 or the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]; the effect of vector-mediated enkephalin on Na(V)1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta-opioid receptor by enkephalin prevents the increase in neuronal Na(V)1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta-opioid receptor and voltage-gated sodium channels.

Expression of Nav1.9 Channels in Human Dental Pulp and Trigeminal Ganglion

There is a higher incidence of local anesthetic failure in endodontic patients experiencing pulpal hyperalgesia. Up-regulation of Nav1.9, a voltage-gated sodium channel isoform, might play a key role in local anesthetic failure because Nav1.9 channels increase neuronal excitability and have low sensitivity to blockade by local anesthetics. Immunocytochemistry was used to examine Nav1.9 channel expression in axons of symptomatic (painful) versus asymptomatic human dental pulp and to determine Nav1.9 expression levels in neuronal somata of the human trigeminal ganglion. Nav1.9 channel immunoreactivity on pulpal axons was significantly increased in painful teeth. Nav1.9 channels were expressed in membranes and cytoplasm of human trigeminal ganglion neurons, with the highest expression in small neuronal somata. Nav1.9 expression in the trigeminal ganglion coupled with increased expression in symptomatic pulp might contribute to hypersensitivity of inflamed pulps and local anesthetic failure. Furthermore, the present study suggests that Nav1.9 channels are potential targets for novel anesthetics.

Painful Research: Identification of a Small-Molecule Inhibitor that Selectively Targets Nav1.8 Sodium Channels

Voltage-gated sodium channels in nociceptive neurons are attractive targets for novel pain therapeutics. Although drugs that target voltage-gated sodium channels have proven value as pain therapeutics, the drugs that are currently available are non-specific sodium channel inhibitors, which limit their usefulness. Recently, a selective small-molecule inhibitor of Na(v)1.8, a voltage-gated sodium channel isoform that participates in peripheral pain mechanisms, has been developed. This exciting new compound shows efficacy in several animal models of pain and is anticipated to be only the first of many new isoform-specific sodium channel blockers.

Recombinase‐mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels

SCN5A encodes the predominant voltage-gated sodium channel isoform in human heart and nearly 100 variants have now been described and studied in vitro. However, development of animal models to analyze function of such large numbers of human gene variants represents a continuing challenge in translational medicine. Here, we describe the implementation of a two stage procedure, recombinase-mediated cassette exchange (RMCE), to efficiently and rapidly generate mice in which a full-length human cDNA replaces expression of the murine ortholog. In the first step of RMCE, conventional homologous recombination in mouse ES cells was used to replace scn5a exon 2 (that contains the translation start site) with a cassette acceptor that includes the thymidine kinase gene, flanked by loxP/inverted loxP sites. In the second step, the cassette acceptor site was replaced by the full-length wild-type human SCN5A cDNA by Cre/loxP-mediated recombination. The exchange event occurred in 7/29 (24%) colonies, and the time from electroporation to first homozygotes was only 8 months. PCR-restriction fragment length polymorphism (RFLP) showed that the murine isoform was replaced by the human one, and functional studies indicated that mice with human cardiac sodium channels have wild-type sodium current density, action potential durations, heart rates, and QRS durations. These data demonstrate that RMCE can be used to generate mice in which a targeted allele can be rapidly and efficiently replaced by variants of choice, and thereby can serve as an enabling approach for the functional characterization of ion channel and other DNA variants.

Modeling the pore structure of voltage-gated sodium channels in closed, open, and fast-inactivated conformation reveals details of site 1 toxin and local anesthetic binding

In this work molecular modeling was applied to generate homology models of the pore region of the Na(v)1.2 and Na(v)1.8 isoforms of human voltage-gated sodium channels. The models represent the channels in the resting, open, and fast-inactivated states. The transmembrane portions of the channels were based on the equivalent domains of the closed and open conformation potassium channels KcsA and MthK, respectively. The critical selectivity loops were modeled using a structural template identified by a novel 3D-search technique and subsequently merged with the transmembrane portions. The resulting draft models were used to study the differences of tetrodotoxin binding to the tetrodotoxin-sensitive Na(v)1.2 (EC50: 0.012 microM) and -insensitive Na(v)1.8 (EC50: 60 microM) isoforms, respectively. Furthermore, we investigated binding of the local anesthetic tetracaine to Na(v)1.8 (EC50: 12.5 microM) in resting, conducting, and fast-inactivated state. In accordance with experimental mutagenesis studies, computational docking of tetrodotoxin and tetracaine provided (1) a description of site 1 toxin and local anesthetic binding sites in voltage-gated sodium channels. (2) A rationale for site 1 toxin-sensitivity versus -insensitivity in atomic detail involving interactions of the Na(v)1.2 residues F385-I and W943-II. (3) A working hypothesis of interactions between Na(v)1.8 in different conformational states and the local anesthetic tetracaine.

Use-Dependent Potentiation of the Na v1.6 Sodium Channel

Na v1.2 and Na v1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Na v1.2 and Na v1.6 in response to a rapid series of repetitive depolarizations. Currents through Na v1.6 coexpressed with β1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Na v1.2 with β1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Na v1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Na v1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Na v1.6. These results indicate that potentiation of Na v1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Na v1.2. The data suggest that Na v1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Na v1.2.

Jingzhaotoxin-III, a Novel Spider Toxin Inhibiting Activation of Voltage-gated Sodium Channel in Rat Cardiac Myocytes

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

Channel activation voltage alone is directly altered in an isoform-specific manner by Na(v1.4) and Na(v1.5) cytoplasmic linkers.

The isoform-specific direct role of cytoplasmic loops in the gating of two voltage-gated sodium channel isoforms, the human cardiac channel (Na(v1.5); hH1) and the human adult skeletal muscle channel (Na(v1.4); hSkM1), was investigated. Comparison of biophysical characteristics was made among hSkM1, hH1, and several hSkM1/hH1 chimeras in which the putative cytoplasmic loops that join domain I to II (loop A) and domain II to III (loop B) from one isoform replaced one or both of the analogous loops from the other isoform. For all parameters measured, hSkM1 and hH1 behavior were significantly different. Comparison of hSkM1 and hH1 biophysical characteristics with the function of their respective chimeras indicate that only the half-activation voltage ( V(a)) is directly and differently altered by the species of cytoplasmic loop such that a channel consisting of one or both hSkM1 loops activates at smaller depolarizations, while a larger depolarization is required for activation of a channel containing one or both of the analogous hH1 loops. When either cardiac channel loop A or B is attached to hSkM1, a 6-7 mV depolarizing shift in V(a) is measured, increasing to a nearly 20 mV depolarization when both cardiac-channel loops are attached. The addition of either skeletal muscle-channel loop to hH1 causes a 7 mV hyperpolarization in V(a), which increases to about 10 mV for the double loop chimera. There is no significant difference in either steady-state inactivation or in the recovery from inactivation data between hSkM1 and its chimeras and between hH1 and its chimeras. Data indicate that the cytoplasmic loops contribute directly to the magnitude of the window current, suggesting that channels containing skeletal muscle loops have three times the peak persistent channel activity compared to channels containing the cardiac loops. An electrostatic mechanism, in which surface charge differences among these loops might alter differently the voltage sensed by the gating mechanism of the channel, can not account for the observed isoform-specific effects of these loops only on channel activation voltage. In summary, although the DI-DII and DII-DIII loop structures among isoforms are not well conserved, these data indicate that only one gating parameter, V(a) is affected directly and in an isoform-specific manner by these divergent loop structures, creating loop-specific window currents and percentages of persistently active channels at physiological voltages that will likely impact the excitability of the cell.