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The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 NTD: Y87C–here newly identified–, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80–105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.

Aim The voltage‐gated sodium channel NaV1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5amutations. We investigated when sodium current (INa) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV1.5 dysfunction impacts on cardiac development. Methods Involvement of NaV1.5‐generated INa in murine cardiac electrical function was assessed by optical mapping in wild type (WT) embryos (embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µmol/L). INa was assessed by patch‐clamp analysis in cardiomyocytes isolated from WT embryos (E9.5‐17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a‐1798insD−/− embryos (E9.5‐10.5) and their WT littermates. Results In WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch‐clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a‐1798insD−/− embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a‐1798insD−/− E9.5 embryos before the onset of INa, in addition to reduced cardiac tissue volume compared to WT littermates. Conclusion We here demonstrate that NaV1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a‐1798insD−/− embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV1.5 dysfunction impacts on embryonic cardiac development in a non‐electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV1.5 dysfunction.

Supplemental Digital Content is Available in the Text. Severe acute respiratory syndrome coronavirus 2's spike protein promotes analgesia by interfering with vascular endothelial growth factor-A/NRP1 pathway, which may affect disease transmission dynamics., Global spread of severe acute respiratory syndrome coronavirus 2 continues unabated. Binding of severe acute respiratory syndrome coronavirus 2's spike protein to host angiotensin-converting enzyme 2 triggers viral entry, but other proteins may participate, including the neuropilin-1 receptor (NRP-1). Because both spike protein and vascular endothelial growth factor-A (VEGF-A)—a pronociceptive and angiogenic factor, bind NRP-1, we tested whether spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuron firing was blocked by spike protein and NRP-1 inhibitor EG00229. Pronociceptive behaviors of VEGF-A were similarly blocked through suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A “silencing” of pain through subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.

Proliferating cells, typically considered “nonexcitable,” nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.

// Harry J. Gould III 1, 3, 5, 6, 7 , Jack Norleans 2 , T. David Ward 4 , Chasiti Reid 2 and Dennis Paul 1, 2, 3, 5, 6, 7 1 Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 2 Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA, USA 3 Department of Anesthesiology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 4 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, LA, USA 5 Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA 6 Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA, USA 7 Center of Excellence for Oral and Craniofacial Biology, Louisiana State University Health Sciences Center, New Orleans, LA, USA Correspondence to: Harry J. Gould III, email: hgould@lsuhsc.edu Dennis Paul, email: dpaul@lsuhsc.edu Keywords: targeted osmotic lysis; epithelial carcinoma; cancer; sodium channels; sodium pumps Received: October 29, 2017 Accepted: February 21, 2018 Epub: February 26, 2018 Published: March 20, 2018 ABSTRACT Sodium influx through voltage-gated sodium channels (VGSCs) coupled with balanced removal of sodium ions via Na + , K + -ATPase is a major determinant of cellular homeostasis and intracellular ionic concentration. Interestingly, many metastatic carcinomas express high levels of these channels. We hypothesized that if excess VGSCs are activated and Na + , K + -ATPase is simultaneously blocked, the intracellular Na + concentration should increase, resulting in water movement into the cell, causing swelling and lytic cell death. MDA-MB-231 breast cancer cells over-express VGSCs by 7-fold. To test our hypothesis, we treated these cells in vitro with the Na + , K + -ATPase blocker, ouabain, and then stimulated with a sublethal electric current. For in vivo histologic and survival studies, MDA-MB-231 xenografts were established in Nu/J mice. Mice injected with saline or ouabain were electrically stimulated with trains of 10 msec 10V DC pulses. Within seconds to minutes, the cells swelled and lysed. MCF-10a cells, which express normal VGSCs levels, were unaffected by this treatment. Cells from the weakly-malignant cell line, MCF-7, which express 3-fold greater VGSCs than MCF-10a cells, displayed an intermediate time-to-lysis. The rate of lysis correlated directly with the degree of sodium channel expression and malignancy. We also demonstrated efficacy in cell lines from prostate, colon and lung carcinomas. Treated MDA-MB-231 xenografts showed 60–80% cell death. In survival studies, TOL-treated mice showed significantly slower tumor growth vs. controls. These results are evidence that this ”targeted osmotic lysis” represents a novel method for selectively killing cancer cells and warrants further investigation as a potential treatment for advanced and end-stage breast cancer.

Key points Auditory brainstem neurons of all vertebrates fire phase‐locked action potentials (APs) at high rates with remarkable fidelity, a process controlled by specialized anatomical and biophysical properties.This is especially true in the avian nucleus magnocellularis (NM) – the analogue of the mammalian anteroventral cochlear nucleus.In addition to high voltage‐activated potassium (KHVA) channels, we report, using whole cell physiology and modelling, that resurgent sodium current (I NaR) of sodium channels (NaV) is equally important and operates synergistically with KHVA channels to enable rapid AP firing in NM.Anatomically, we detected strong NaV1.6 expression near hearing maturation, which was less distinct during hearing development despite functional evidence of I NaR, suggesting that multiple NaV channel subtypes may contribute to I NaR.We conclude that I NaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing. Abstract Auditory brainstem neurons are functionally primed to fire action potentials (APs) at markedly high rates in order to rapidly encode the acoustic information of sound. This specialization is critical for survival and the comprehension of behaviourally relevant communication functions, including sound localization and distinguishing speech from noise. Here, we investigated underlying ion channel mechanisms essential for high‐rate AP firing in neurons of the chicken nucleus magnocellularis (NM) – the avian analogue of bushy cells of the mammalian anteroventral cochlear nucleus. In addition to the established function of high voltage‐activated potassium channels, we found that resurgent sodium current (I NaR) plays a role in regulating rapid firing activity of late‐developing (embryonic (E) days 19–21) NM neurons. I NaR of late‐developing NM neurons showed similar properties to mammalian neurons in that its unique mechanism of an ‘open channel block state’ facilitated the recovery and increased the availability of sodium (NaV) channels after depolarization. Using a computational model of NM neurons, we demonstrated that removal of I NaR reduced high‐rate AP firing. We found weak I NaR during a prehearing period (E11–12), which transformed to resemble late‐developing I NaR properties around hearing onset (E14–16). Anatomically, we detected strong NaV1.6 expression near maturation, which became increasingly less distinct at hearing onset and prehearing periods, suggesting that multiple NaV channel subtypes may contribute to I NaR during development. We conclude that I NaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing., Key points Auditory brainstem neurons of all vertebrates fire phase‐locked action potentials (APs) at high rates with remarkable fidelity, a process controlled by specialized anatomical and biophysical properties.This is especially true in the avian nucleus magnocellularis (NM) – the analogue of the mammalian anteroventral cochlear nucleus.In addition to high voltage‐activated potassium (KHVA) channels, we report, using whole cell physiology and modelling, that resurgent sodium current (I NaR) of sodium channels (NaV) is equally important and operates synergistically with KHVA channels to enable rapid AP firing in NM.Anatomically, we detected strong NaV1.6 expression near hearing maturation, which was less distinct during hearing development despite functional evidence of I NaR, suggesting that multiple NaV channel subtypes may contribute to I NaR.We conclude that I NaR plays an important role in regulating rapid AP firing for NM neurons, a property that may be evolutionarily conserved for functions related to similar avian and mammalian hearing.

Previously, it was found that 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP-315) effectively protects mice from maximal electroshock-induced seizures. The aim of this study was to determine possible interactions between TP-315 and different molecular targets, i.e. GABAA receptors, voltage-gated sodium channels, and human neuronal α7 and α4β2 nicotinic acetylcholine receptors. The influence of TP-315 on the viability of human hepatic HepG2 cells was also established using PrestoBlue and ToxiLight assays. It was found that the anticonvulsant activity of TP-315 results (at least partially) from its influence on voltage-gated sodium channels (VGSCs). Moreover, the title compound slightly affected the viability of human hepatic cells.

Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena.

Key points The generation of dendritic spikes and the consequent sharp tuning of neuronal responses are together attainable even when iso‐feature synapses are randomly dispersed across the dendritic arbor.Disparate combinations of channel conductances with distinct configurations of randomly dispersed place field synapses concomitantly yield similar sharp tuning profiles and similar functional maps of several intrinsic properties.Targeted synaptic plasticity converts silent cells to place cells for specific place fields in models with disparate channel combinations that receive dispersed synaptic inputs from multiple place field locations.Dispersed localization of iso‐feature synapses is a strong candidate for achieving sharp feature selectivity in neurons across sensory‐perceptual systems, with several degrees of freedom in relation to synaptic locations.Quantitative evidence for the possibility that degeneracy (i.e. the ability of disparate structural components to yield similar functional outcomes) could act as a broad framework that effectively accomplishes the twin goals of input‐feature encoding and homeostasis of intrinsic properties without cross interferences. Abstract A prominent hypothesis spanning several sensory‐perceptual systems implicates spatially clustered synapses in the generation of dendritic spikes that mediate sharply‐tuned neuronal responses to input features. In this conductance‐based morphologically‐precise computational study, we tested this hypothesis by systematically analysing the impact of distinct synaptic and channel localization profiles on sharpness of spatial tuning in hippocampal pyramidal neurons. We found that the generation of dendritic spikes, the emergence of an excitatory ramp in somatic voltage responses, the expression of several intrinsic somatodendritic functional maps and sharp tuning of place‐cell responses were all attainable even when iso‐feature synapses are randomly dispersed across the dendritic arbor of models with disparate channel combinations. Strikingly, the generation and propagation of dendritic spikes, reliant on dendritic sodium channels and N‐methyl‐d‐asparate receptors, mediated the sharpness of spatial tuning achieved with dispersed synaptic localization. To ensure that our results were not artefacts of narrow parametric choices, we confirmed these conclusions with independent multiparametric stochastic search algorithms spanning thousands of unique models for each synaptic localization scenario. Next, employing virtual knockout models, we demonstrated a vital role for dendritically expressed voltage‐gated ion channels, especially the transient potassium channels, in maintaining sharpness of place‐cell tuning. Importantly, we established that synaptic potentiation targeted to afferents from one specific place field was sufficient to impart place field selectivity even when intrinsically disparate neurons received randomly dispersed afferents from multiple place field locations. Our results provide quantitative evidence for disparate combinations of channel and synaptic localization profiles to concomitantly yield similar tuning and similar intrinsic properties., Key points The generation of dendritic spikes and the consequent sharp tuning of neuronal responses are together attainable even when iso‐feature synapses are randomly dispersed across the dendritic arbor.Disparate combinations of channel conductances with distinct configurations of randomly dispersed place field synapses concomitantly yield similar sharp tuning profiles and similar functional maps of several intrinsic properties.Targeted synaptic plasticity converts silent cells to place cells for specific place fields in models with disparate channel combinations that receive dispersed synaptic inputs from multiple place field locations.Dispersed localization of iso‐feature synapses is a strong candidate for achieving sharp feature selectivity in neurons across sensory‐perceptual systems, with several degrees of freedom in relation to synaptic locations.Quantitative evidence for the possibility that degeneracy (i.e. the ability of disparate structural components to yield similar functional outcomes) could act as a broad framework that effectively accomplishes the twin goals of input‐feature encoding and homeostasis of intrinsic properties without cross interferences.

Aryl sulfonamide Nav 1.3 or Nav 1.7 voltage-gated sodium (Nav ) channel inhibitors interact with the Domain 4 voltage sensor domain (D4 VSD). During studies to better understand the structure-activity relationship of this interaction, an additional mode of channel modulation, specifically slowing of inactivation, was revealed by addition of a single methyl moiety. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel.Electrophysiology and site-directed mutation were used to compare the effects of PF-06526290 and its desmethyl analogue PF-05661014 on Nav channel function.PF-05661014 selectively inhibits Nav 1.3 versus Nav 1.7 currents by stabilizing inactivated channels via interaction with D4 VSD. In contrast, PF-06526290, which differs from PF-05661014 by a single methyl group, exhibits a dual effect. It greatly slows inactivation of Nav channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Nav subtype selective inhibitor similar to PF-05661014. Mutation of the D4 VSD modulates inhibition of Nav 1.3 or Nav 1.7 by both PF-05661014 and PF-06526290, but has no effect on the inactivation slowing produced by PF-06526290. This finding, along with the absence of functional inhibition of PF-06526290-induced inactivation slowing by PF-05661014, suggests that distinct interactions underlie the two modes of Nav channel modulation.Addition of a methyl group to a Nav channel inhibitor introduces an additional mode of gating modulation, implying that a single compound can affect sodium channel function in multiple ways.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.

Enhanced late NaThe effects of GS-6615 to inhibit late IGS-6615 inhibited ATX-II enhanced late IGS-6615 was a selective inhibitor of late I

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (KD, 1-10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -60 mV, and peaked at -30 and -10 mV, with a current density of 22 +/- 3 and 18 +/- 1 pA pF(-1), respectively. TTX-sensitive iNa inactivated at more positive potentials as compared to TTX-resistant iNa. Using action potential clamp, TTX-sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Nav1.5, and the TTX-sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX-sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 microm) of TTX, which block TTX-resistant iNa as well as TTX-sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

1. We studied the effects of benzylalcohol on heterologously expressed wild type (WT), paramyotonia congenita (R1448H) and hyperkalaemic periodic paralysis (M1360V) mutant alpha-subunits of human skeletal muscle sodium channels. 2. Benzylalcohol blocked rested channels at -150 mV membrane potential, with an ECR(50) of 5.3 mM in wild type, 5.1 mM in R1448H, and 6.2 mM in M1360V. When blockade was assessed at -100 mV, the ECR(50) was reduced in R1448H (2 mM) compared with both wild type (4.3 mM; P

Alveolar epithelial type II pneumocytes were isolated and purified from adult rat lung by elastase digestion and differential adhesion, and cultured in serum-free medium for ∼2 days on glass coverslips for subsequent patch-clamp studies employing symmetrical sodium isethionate solutions. Whole-cell Na+ currents exhibited essentially linear current-voltage relationships which were mildly inhibited (by ∼25 %) by 10 μm amiloride. In contrast, 1 mm Zn2+ inhibited the currents by ∼55 % with an IC50 of ∼134 μm and maximal blockade achieved between 5 and 10 mm. The effects of Zn2+ and amiloride were additive, and independent of the order of blocker addition. Gd2+, Zn2+ and La3+ at 10 mm were all effective at rapidly, reversibly and significantly blocking the amiloride-insensitive currents by ∼60 %. In contrast, Ni2+ was a very weak inhibitor (30 % inhibition at 10 mm). Pimozide (10 μm) caused inhibition of whole-cell cation conductance by ∼55 %. The inhibitory effect of pimozide was concentration dependent with an IC50 of ∼1 μm and was maximally effective between 10 and 30 μm. Sequential addition of Zn2+ and pimozide, in either order, revealed no overlapping inhibitory effect on the amiloride-insensitive conductance, and supported the notion that the Zn2+- and pimozide-sensitive currents are identical. The amiloride-insensitive, Zn2+-blockable conductance was characterised by a Na+/K+ permeability ratio (PNa/PK) of 0.73 ± 0.02. 8Br-cGMP (100 μm), a membrane-permeable analogue of cGMP, evoked a robust activation of whole-cell cation conductance to 220 % of control. This activation was apparent in either the absence or the presence of 10 μm amiloride, but was completely abolished in the presence of Zn2+. These data support the in vivo and in situ observations of a substantial amiloride-resistant Na+ conductance, demonstrate directly that cyclic nucleotide-gated non-selective cation channels are functionally expressed in alveolar epithelial type II cells, and suggest that these channels may contribute to the fluid-reabsorptive driving force in adult lung. It is well accepted that fluid reabsorption from the term lung at birth is inhibited by luminal amiloride (Olver et al. 1986; O'Brodovich et al. 1990), suggesting that amiloride-sensitive Na+ channels may be rate-limiting in the process. The observation that transgenic mice which lack the epithelial sodium channel α-subunit, αENaC, have fatally impaired lung fluid clearance (Hummler et al. 1996) further supports an important role for amiloride-sensitive pathways in the perinatal period. However, in the mature lung, amiloride-insensitive fluid reabsorption accounts for up to 70 % of the total clearance, with some variation among species (Ramsden et al. 1992; Norlin et al. 1998). Even though numerous studies have also reported large amiloride-insensitive whole-cell Na+ currents in adult alveolar epithelial type II cells (e.g. Haskell et al. 1994; Kemp et al. 1997), none has characterised this component and there still appears to be common consent that ENaC activity primarily underlies adult lung fluid homeostasis. That the cellular and in vivo/in situ data both support a role for amiloride-insensitive vectorial Na+ transport highlights the importance of investigating the nature of this component before the precise mechanism by which postnatal fluid homeostasis is maintained can be fully understood. Recently, compelling direct evidence has been presented on the nature of this significant amiloride-insensitive component to adult lung fluid reabsorption. Junor et al. (1999), employing the well-characterised postnatal sheep in situ lung preparation, have convincingly demonstrated that a substantial component of adult lung fluid reabsorption is insensitive to the blocking effects of maximal concentrations of amiloride and that the remaining absorptive response is inhibited by either dichlorobenzamil or pimozide. Both of these latter drugs are relatively selective inhibitors of the cyclic nucleotide-gated non-selective cation channel. Interestingly, stimulated fetal sheep fluid reabsorption is unaffected by similar pharmacological manipulation (Junor et al. 2000), indicating that expression of this channel in the pulmonary epithelium may be developmentally regulated. Cyclic nucleotide-gated cation channels were first described by Fesenko et al. (1985), and were subsequently cloned from retinal rods (Kaupp et al. 1989). The functional channel is heteromeric with a smaller six transmembrane domain α-subunit representing the conductance (Bonigk et al. 1993) and a larger modulatory β-subunit (Korschen et al. 1995). Since the initial molecular characterisation of the bovine rod photoreceptor channel (CNG1), a number of homologues of α-subunits have been cloned by homology screening and PCR, including rat olfactory receptor CNG2 (Dhallan et al. 1990) and bovine cone photoreceptor CNG3 (Biel et al. 1994). Cloned β-subunits include CNG4 (Biel et al. 1996) from cattle, CNG5 (Bradley et al. 1994) from rat and CNG6 from mouse (Gerstner et al. 2000). It is now known that CNG genes are widely expressed in a tissue-specific manner. Their mRNAs have been localised in both sensory and non-sensory tissues, including lung in general (Ding et al. 1997) and airway specifically (Schwiebert et al. 1997), while mRNA for all three α-subunits (CNG1–3) and βCNG are expressed in the pulmonary epithelial cell line A549 (Xu et al. 1999). Expressed cyclic nucleotide-gated channels have a number of pharmacological and biophysical characteristics that distinguish them from ENaC or other amiloride-sensitive Na+ channels. As employed in the postnatal lung reabsorption study, they are selectively blocked by the dopamine D2 receptor antagonist pimozide (Junor et al. 1999). This is an important pharmacological tool since adult rat alveolar Na+ transport is not affected by another D2 receptor antagonist, S-sulpiride (Barnard et al. 1999), suggesting that pimozide action in lung is due to binding to cyclic nucleotide-gated cation channels. Characteristically, these channels are activated by micromolar concentrations of cGMP, or its membrane-permeable analogue 8Br-cGMP (Fesenko et al. 1985), are inhibited by di- and trivalent cations (Karpen et al. 1993), and show little or no preference for Na+ over K+ as the permeating ion, with GK often very slightly higher than GNa (Picones & Korenbrot, 1992). In the present paper, we have employed the patch-clamp technique to study amiloride-insensitive whole-cell cation currents and their regulation by cGMP, inorganic cations and pimozide in order to address the hypothesis that adult alveolar epithelial type II cells functionally express cyclic nucleotide-gated cation channels. Some of this work has appeared previously in abstract form (Kemp et al. 1998).

BACKGROUND AND PURPOSE Voltage-gated sodium channels (Na(V) channels) are key players in the generation and propagation of action potentials, and selective blockade of these channels is a promising strategy for clinically useful suppression of electrical activity. The conotoxin µ-CnIIIC from the cone snail Conus consors exhibits myorelaxing activity in rodents through specific blockade of skeletal muscle (Na(V) 1.4) Na(V) channels. EXPERIMENTAL APPROACH We investigated the activity of µ-CnIIIC on human Na(V) channels and characterized its inhibitory mechanism, as well as the molecular basis, for its channel specificity. KEY RESULTS Similar to rat paralogs, human Na(V) 1.4 and Na(V) 1.2 were potently blocked by µ-CnIIIC, the sensitivity of Na(V) 1.7 was intermediate, and Na(V) 1.5 and Na(V) 1.8 were insensitive. Half-channel chimeras revealed that determinants for the insensitivity of Na(V) 1.8 must reside in both the first and second halves of the channel, while those for Na(V) 1.5 are restricted to domains I and II. Furthermore, domain I pore loop affected the total block and therefore harbours the major determinants for the subtype specificity. Domain II pore loop only affected the kinetics of toxin binding and dissociation. Blockade by µ-CnIIIC of Na(V) 1.4 was virtually irreversible but left a residual current of about 5%, reflecting a 'leaky' block; therefore, Na(+) ions still passed through µ-CnIIIC-occupied Na(V) 1.4 to some extent. TTX was excluded from this binding site but was trapped inside the pore by µ-CnIIIC. CONCLUSION AND IMPLICATIONS Of clinical significance, µ-CnIIIC is a potent and persistent blocker of human skeletal muscle Na(V) 1.4 that does not affect activity of cardiac Na(V) 1.5.

Cystic fibrosis is a genetic condition caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Loss or altered function of the product of this gene, the CFTR protein, affects the transport of ions across cell membranes in several tissues in the body which in turn, through mechanisms which are still not fully understood, results in the disease.

Aberrant protein glycosylation plays major roles in neurodegenerative diseases, including Parkinson's disease (PD). Glycoproteomics showed that the glycosylation of sodium channel β4 was significantly increased in human brain tissue. β4-specific antibodies reacted in immunoblot assays with the 35- and 38-kDa bands from the membrane fractions isolated from neonatal PD transgenic mice but only with the 35-kDa band of the neonatal wild-type mice. The size of the 38-kDa immunoreactive protein is in close agreement with previously reported, suggesting heavy glycosylation of this protein in adult wild-type and neonatal PD transgenic brain tissues. However, the neonatal wild-type mice membrane fractions only contained the 35-kDa immunoreactive protein, and the additional 38-kDa band was not shown until postnatal day 7. Enzymatic deglycosylation of the membrane preparations only converted the 38-kDa band into a faster migrating protein, which was consistent with heavy glycosylation of this protein. The glycosylated state of β4 was developmentally regulated and was altered in disease state. Neurite outgrowth assay demonstrated that overexpression of deglycosylated mutant β4-MUT accelerated neurite extension and increased the number of filopodia-like protrusions, when compared with β4-WT and the vector. These results suggest that extensive glycosylation of β4 subunit play roles in morphological changes, and the altered glycosylation may be involved in the pathogenesis of PD.

Rat prostate cancer cells have been previously investigated using two cell lines: a highly metastatic one (Mat-Ly-Lu) and a nonmetastatic one (AT-2). It turns out that the highly metastatic Mat-Ly-Lu cells exhibit a phenomenon of cathodal galvanotaxis in an electric field which can be blocked by interrupting the voltage-gated sodium channel (VGSC) activity. The VGSC activity is postulated to be characteristic for metastatic cells and seems to be a reasonable driving force for motile behavior. However, the classical theory of cellular motion depends on calcium ions rather than sodium ions. The current research provides a theoretical connection between cellular sodium inflow and cathodal galvanotaxis of Mat-Ly-Lu cells. Electrical repulsion of intracellular calcium ions by entering sodium ions is proposed after depolarization starting from the cathodal side. The disturbance in the calcium distribution may then drive actin polymerization and myosin contraction. The presented modeling is done within a continuous one-dimensional Poisson-Nernst-Planck equation framework.

Interstitial cells of Cajal (ICCs) are the pacemaking cells in the gastrointestinal muscles that generate the rhythmic oscillations in membrane potential known as slow waves. ICCs also mediate or transduce inputs from the enteric nervous system. Substance P (SubP) is a member of the family of mammalian tachykinin peptides that are predominantly released by enteric neurons. This study assessed the relationship of Na(+)-leak channel (NALCN) in the SubP-induced depolarization in pacemaking activity in the gastrointestinal tract. The patch-clamp technique for whole-cell recording was used in cultured cluster and single ICCs. Electrophysiological and pharmacological properties of SubP in ICC pacemaking activity were similar to those of NALCN. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of NALCN messenger RNA and protein in mouse small intestine. NALCN is involved in the SubP-induced depolarization of intestinal pacemaking activity. The protein is a potential target for pharmacological treatment of motor disorders of the gut.

Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.

Five BC1 lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.

Low current cortex stimulation produces a sparse and distributed set of activated cells often with distances of several hundred micrometers between cell bodies and the microelectrode. A modeling study based on recently measured densities of high threshold sodium channels Nav1.2 in dendrites and soma and low threshold sodium channels Nav1.6 in the axon shall identify spike initiation sites including a discussion on dendritic spikes. Varying excitability along the neural axis has been observed while studying different electrode positions and configurations. Although the axon initial segment (AIS) and nodes of Ranvier are most excitable, many thin axons and dendrites which are likely to be close to the electrode in the densely packed cortical regions are also proper candidates for spike initiation sites. Cathodic threshold ratio for thin axons and dendrites is about 1:3, whereas 0.2 μm diameter axons passing the electrode tip in 10 μm distance can be activated by 100 μs pulses with 2.6 μA. Direct cathodic excitation of dendrites requires a minimum electrode-fiber distance, which increases with dendrite diameter. Therefore thin dendrites can profit from the stronger electrical field close to the electrode but low current stimulation cannot activate large diameter dendrites, contrary to the inverse recruitment order known from peripheral nerve stimulation. When local depolarization fails to generate a dendritic spike, stimulation is possible via intracellular current flow that initiates an action potential, for example 200 μm distant in the low threshold AIS or in certain cases at the distal dendrite ending. Beside these exceptions, spike initiation site for cathodic low current stimulation appears rather close to the electrode.

Background and purpose: The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)-sensitive voltage-gated Na+ channels were investigated in mouse vas deferens. Experimental approach: Effects of veratridine on TTX-sensitive Na+ currents (INa) in vas deferens myocytes dispersed from BALB/c mice, homozygous mice with a null allele of NaV1.6 (NaV1.6−/−) and wild-type mice (NaV1.6+/+) were studied using patch-clamp techniques. Tension measurements were also performed to compare the effects of veratridine on phasic contractions in intact tissues. Key results: In whole-cell configuration, veratridine had a concentration-dependent dual action on the peak amplitude of INa: INa was enhanced by veratridine (1–10 µM), while higher concentrations (≥30 µM) inhibited INa. Additionally, two membrane current components were evoked by veratridine, namely a sustained inward current during the duration of the depolarizing rectangular pulse and a tail current at the repolarization. Although veratridine caused little shift of the voltage dependence of the steady-state inactivation curve and the activation curve for INa, veratridine enhanced a non-inactivating component of INa. Veratridine caused no detectable contractions in vas deferens from NaV1.6−/− mice, although in tissues from NaV1.6+/+ mice, veratridine (≥3 µM) induced TTX-sensitive contractions. Similarly, no detectable inward currents were evoked by veratridine in NaV1.6−/− vas deferens myocytes, while veratridine elicited both the sustained and tail currents in cells taken from NaV1.6+/+ mice. Conclusions and implications: These results suggest that veratridine possesses a dual action on INa and that the veratridine-induced activation of contraction is induced by the activation of NaV1.6 channels.

Mandarin translation of abstract Background and purpose: The voltage-gated Na+ channels (Nav) and their corresponding current (INa) are involved in several cellular processes, crucial to metastasis of cancer cells. We investigated the effects of eicosapentaenoic (EPA), an omega-3 polyunsaturated fatty acid, on INa and metastatic functions (cell proliferation, endocytosis and invasion) in human and rat prostate cancer cell lines (PC-3 and Mat-LyLu cells). Experimental approach: The whole-cell voltage clamp technique and conventional/quantitative real-time reverse transcriptase polymerase chain reaction analysis were used. The presence of Nav proteins was shown by immunohistochemical methods. Alterations in the fatty acid composition of phospholipids after treatment with EPA and metastatic functions were also examined. Key results: A transient inward Na+ current (INa), highly sensitive to tetrodotoxin, and NaV proteins were found in these cells. Expression of NaV1.6 and NaV1.7 transcripts (SCN8A and SCN9A) was predominant in PC-3 cells, while NaV1.7 transcript (SCN9A) was the major component in Mat-LyLu cells. Tetrodotoxin or synthetic small interfering RNA targeted for SCN8A and SCN9A inhibited metastatic functions (endocytosis and invasion), but failed to inhibit proliferation in PC-3 cells. Exposure to EPA produced a rapid and concentration-dependent suppression of INa. In cells chronically treated (up to 72h) with EPA, the EPA content of cell lipids increased time-dependently, while arachidonic acid content decreased. Treatment of PC-3 cells with EPA decreased levels of mRNA for SCN9A and SCN8A, cell proliferation, invasion and endocytosis. Conclusion and implications: Treatment with EPA inhibited INa directly and also indirectly, by down-regulation of Nav mRNA expression in prostate cancer cells, thus inhibiting their metastatic potential. Mandarin translation of abstract

While some regard the flail arm syndrome as a variant of amyotrophic lateral sclerosis (ALS), others have argued that it is a distinct clinical entity. Consequently, the present study applied novel central and peripheral nerve excitability techniques to further explore disease pathophysiology in flail arm syndrome.Cortical and peripheral nerve excitability studies were undertaken in 11 flail arm patients, defined by muscle weakness limited to the proximal aspects of the upper limbs for at least 24 months.Mean age at disease onset (60.3 years) was similar to other ALS phenotypes (58.3 years), with strong male predominance (male:female distribution: flail arm 10:1; ALS 1.5:1; p0.05) and prolonged disease duration (flail arm 62.5 months; ALS 15.8 months; p0.05). There was evidence of cortical hyperexcitability in flail arm patients, similar to findings in ALS, with reduction in short interval intracortical inhibition (flail arm 0.8 (0.6)%; ALS 4.1 (1.1)%; controls 8.5 (1.0)%; p0.0001) and resting motor threshold (flail arm 53.4 (2.8)%; ALS 56.6 (1.8)%; controls 60.7 (1.5)%; p0.05), along with an increase in motor evoked potential amplitude (flail arm 49.5 (9.0)%; ALS 44.4 (4.9)%; controls 25.8 (2.8)%; p0.05). Peripheral nerve excitability studies demonstrated changes consistent with upregulation in persistent Na+ currents and reduction of slow K+ conductances, similar to findings in ALS.This study has demonstrated the presence of cortical hyperexcitability in flail arm syndrome, along with abnormalities in peripheral nerve excitability, findings consistent with previous studies in other ALS phenotypes. By demonstrating the presence of upper motor neuron dysfunction, the present study suggests that the flail arm syndrome is an unusual variant of ALS.

The ventricular action potential plateau is a phase of high resistance, which makes ventricular myocytes vulnerable to small electrical perturbations. We developed a computationally based model of GS-458967 interaction with the cardiac Na+ channel, informed by experimental data recorded from guinea pig isolated single ventricular myocytes. The model predicts that the therapeutic potential of GS-458967 derives largely from the designed property of significant potent selectivity for INaL.Selective inhibition of the slowly inactivating or late Na(+) current (INaL) in patients with inherited or acquired arrhythmia syndrome may confer therapeutic benefit by reducing the incidence of triggers for arrhythmia and suppressing one component of arrhythmia-promoting cardiac substrates (e.g. prolonged refractoriness and spatiotemporal dispersion of action potential duration). Recently, a novel compound that preferentially and potently reduces INaL, GS-458967 (IC50 for block of INaL = 130 nM) has been studied. Experimental measurements of the effects of GS-458967 on endogenous INaL in guinea pig ventricular myocytes demonstrate a robust concentration-dependent reduction in action potential duration (APD). Using experimental data to calibrate INaL and the rapidly activating delayed rectifier K(+) current, IKr, in the Faber-Rudy computationally based model of the guinea pig ventricular action potential, we simulated effects of GS-458967 on guinea pig ventricular APD. GS-458967 (0.1 μM) caused a 28.67% block of INaL and 12.57% APD shortening in experiments, while the model predicted 10.06% APD shortening with 29.33% block of INaL. An additional effect of INaL block is to reduce the time during which the membrane potential is in a high resistance state (i.e. the action potential plateau). To test the hypothesis that targeted block of INaL would make ventricular myocytes less susceptible to small electrical perturbations, we used the computational model to test the degree of APD prolongation induced by small electrical perturbations in normal cells and in cells with simulated long QT syndrome. The model predicted a substantial dose-dependent reduction in sensitivity to small electrical perturbations as evidenced by action potential duration at 90% repolarization variability in the presence of GS-458967-induced INaL block. This effect was especially potent in the 'disease setting' of inherited long QT syndrome. Using a combined experimental and theoretical approach, our results suggest that INaL block is a potent therapeutic strategy. This is because reduction of INaL stabilizes the action potential waveform by reducing depolarizing current during the plateau phase of the action potential. This reduces the most vulnerable phase of the action potential with high membrane resistance. In summary, by reducing the sensitivity of the myocardial substrate to small electrical perturbations that promote arrhythmia triggers, agents such as GS-458967 may constitute an effective antiarrhythmic pharmacological strategy.

The properties of chorioallantoic membrane derived from Large White Landrace sows at 45, 65 and 100 days gestation are examined. Under short circuit conditions positive charge flows from fetal to maternal sides of the tissue. Na+ is shown to be the sole charge carrier as the short circuit current is inhibited reversibly by fetal applications of amiloride and replacement of Na+ by choline in the Ringer solution, and irreversibly by both fetal and maternal applications of ouabain. The initial short circuit current is smaller at day 100 compared to days 45 and 65. The dose responses to amiloride indicate that the epithelial sodium channel (ENaC) is involved in the movement of Na+ and that it is accessible on the fetal side of the tissue only. Immunostaining shows that the ENaC-alpha subunit is present in both the allantoic membrane and the trophoblast. Uptake studies using microvillous (apical) membrane vesicles suggest it is either inactive or only weakly active at this site. The trophoblast at day 100 has a higher content of ENaC than at days 45 and 65. This is the first report of the presence of ENaC in placental tissues. The effects of ouabain indicate the presence of a Na+ pump that is more readily inhibited by applications of the drug on the maternal aspect of the tissue than on the fetal side. Differential mechanisms may be present that would allow net movement of Na+ in either direction across the chorioallantoic membrane according to the changing demands of the developing fetus.

Neurotoxins such as aconitine (AC) bind to receptor site 2 on voltage-gated sodium channels and modify channel kinetics. Although AC modification typically induces hyperpolarizing shifts in sodium channel activation, the effects on channel inactivation seem to vary depending on the tissue origin of the channel. In the present study, the alpha subunits of human heart (hH1) and rat skeletal muscle (mu1) sodium channels were transiently expressed in human embryonic kidney (HEK293t) cells. Whole-cell currents were examined before and after AC modification of the channels to determine whether the toxin had isoform-specific effects on channel kinetics. The magnitudes of the hyperpolarizing shifts in steady-state current activation and inactivation were similar for AC-modified hH1 and mu1 channels, and AC modification did not alter the voltage dependence of macroscopic current decay of either channel subtype. There were two notable differences between hH1 and mu1 channels after AC modification. First, the steady-state availability of AC-modified mu1 channels decreased by 5-10% after very negative conditioning pulses. Second, AC-modified mu1 channels inactivated completely at all voltages, whereas AC-modified hH1 channels exhibited sustained inward currents at voltages near the threshold of current activation. Interestingly, AC-modified hH1 channels inactivated completely if the external solution did not contain sodium ions. The data demonstrate that AC modification affects the activation of hH1 and mu1 channels similarly but affects inactivation of the two channels distinctly. The results also imply that the reduced inactivation of AC-modified hH1 channels at least partially depends on the presence of extracellular sodium.

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.In the presence of 1-10 μM AEA, suppression of both Na(+) and L-type Ca(2+) channels was observed. Inhibition of Na(+) channels was voltage and Pertussis toxin (PTX) - independent. Radioligand-binding studies indicated that specific binding of [(3) H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd . Further studies on L-type Ca(2+) channels indicated that AEA potently inhibited these channels (IC50 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba(2+) currents. MetAEA also inhibited Na(+) and L-type Ca(2+) currents. Radioligand studies indicated that specific binding of [(3) H]isradipine, was inhibited significantly by metAEA. (10 μM), changing Bmax but not Kd .Results indicate that AEA inhibited the function of voltage-dependent Na(+) and L-type Ca(2+) channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation.

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD50 (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice., A wheat germ embryo derived cell-free translation system expresses a biologically active, highly toxic scorpion venom protein that is fully neutralized by a camel single domain antibody fragment raised against the native scorpion toxin.

1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.

OBJECTIVES—Patients with paralysis periodica paramyotonica exhibit a clinical syndrome with characteristics of both hyperkalaemic periodic paralysis and paramyotonia congenita. In several types of periodic paralysis associated with hyperkalaemia, mutations in the skeletal muscle sodium channel (SCN4A) gene have been previously reported. Phenotypic variations of mutations in SCN4A, however, have not been described yet. The present study aimed to evaluate genetic variations in a family with clinical and electrophysiological characteristics of paralysis periodica paramyotonia. METHODS—Seven members of a family affected with symptoms of paralysis periodica paramyotonia were studied by electrophysiological and genetic analyses. There were increased serum potassium concentrations in four members during paralytic attacks induced by hyperkalaemic periodic paralysis provocation tests. Short exercise tests before and after cold immersion were carried out in four patients to distinguish electrophysiological characteristics of hyperkalaemic periodic paralysis and paramyotonia. Sequencing analyses of SCN4A were performed on one patient and a normal control to identify polymorphisms. Restriction fragment length polymorphism (RFLP) analysis was then performed at the identified polymorphic sites. RESULTS—Electrophysiological studies showed both exercise sensitivity and temperature sensitivity. Compound motor action potential (CMAP) amplitudes were decreased (7.3%-28.6%) after short exercise tests. The CMAP amplitudes were even more severely decreased (21.7%-56.5%) in short exercise tests after cold exposure. Three polymorphic sites, Gln371Glu, Thr704Met, and Aspl376Asn were identified in SCN4A. RFLP analyses showed that all affected patients carried the Thr704Met mutation, whereas unaffected family members and a normal control did not. CONCLUSION— Phenotypic variation of the Thr704Met mutation, which was previously reported in patients with hyperkalaemic periodic paralysis, is described in a family affected with paralysis periodica paramyotonia.

Although a persistent component of the sodium current (INaP) was described in cardiac tissue about three decades ago, its physiological role and potential as a therapeutic target was not immediately apparent. Subsequent demonstrations that INaP is enhanced by hypoxia and ischaemia, and that Na+ influx via INaP may contribute to cellular damage, diastolic dysfunction and arrhythmias during ischaemia and reperfusion, raised interest in INaP as a target for anti-ischaemic drugs. Several agents have now been developed to clinical stages, which have INaP block as either their main action, or as a useful co-effect. In this issue of the British Journal of Pharmacology, Vacher et al. report the anti-ischaemic actions of F15845, which appears to exhibit the most selective block of INaP yet described. Its efficacy in animal models of angina raises the prospect of new, specific, INaP blockers that may represent a largely unexploited opportunity for a new class of anti-ischaemic compounds.

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective “knock-down” of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.

Although it is well established that hyperexcitability and/or increased baseline sensitivity of primary sensory neurons can lead to abnormal burst activity associated with pain, the underlying molecular mechanisms are not fully understood. Early studies demonstrated that, after injury to their axons, neurons can display changes in excitability, suggesting increased sodium channel expression, and, in fact, abnormal sodium channel accumulation has been observed at the tips of injured axons. We have used an ensemble of molecular, electrophysiological, and pharmacological techniques to ask: what types of sodium channels underlie hyperexcitability of primary sensory neurons after injury? Our studies demonstrate that multiple sodium channels, with distinct electrophysiological properties, are encoded by distinct mRNAs within small dorsal root ganglion (DRG) neurons, which include nociceptive cells. Moreover, several DRG neuron-specific sodium channels now have been cloned and sequenced. After injury to the axons of DRG neurons, there is a dramatic change in sodium channel expression in these cells, with down-regulation of some sodium channel genes and up-regulation of another, previously silent sodium channel gene. This plasticity in sodium channel gene expression is accompanied by electrophysiological changes that poise these cells to fire spontaneously or at inappropriate high frequencies. Changes in sodium channel gene expression also are observed in experimental models of inflammatory pain. Thus, sodium channel expression in DRG neurons is dynamic, changing significantly after injury. Sodium channels within primary sensory neurons may play an important role in the pathophysiology of pain.

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration-dependent manner, with an IC50 of 274 microm (confidence limits: 141-407 microm). The inhibition was complete, voltage-independent, and slowly reversible. Toluene had no effect on: (i). the shape of the I-V curves; (ii). the reversal potential of Na+; and (iii). the steady-state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use- and frequency-dependent. In rat cardiac myocytes, 300 microm toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use- and frequency-dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect.

Voltage-activated Na(+) channels contain one distinct α-subunit. In the brain NaV 1.1, NaV 1.2, NaV 1.3 and NaV 1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na(+) channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine.Na(+) currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits.NaV 1.2 and NaV 1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and NaV 1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to NaV 1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin.The four Na(+) channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na(+) channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development.

Treatment with methadone is associated with severe cardiac arrhythmias, a side effect that seems to result from an inhibition of cardiac hERG K⁺ channels. However, several other opioids are inhibitors of voltage-gated Na⁺ channels. Considering the common assumption that an inhibition of the cardiac Na⁺ channel Na(v)1.5, is the primary mechanism for local anaesthetic (LA)-induced cardiotoxicity, we hypothesized that methadone has LA-like properties leading to a modulation of Na(v)1.5 channels.The whole-cell patch clamp technique was applied to investigate the effects of methadone on wild-type and mutant human Na(v)1.5 channels expressed in HEK293 cells. A homology model of human Na(v)1.5 channels was used to perform automated ligand-docking studies.Methadone inhibited Na(v)1.5 channels in a state-dependent manner, that is, tonic block was stronger with inactivated channels than with resting channels and a use-dependent block at 10 Hz. Methadone induced a concentration-dependent shift of the voltage dependency of both fast and slow inactivation towards more hyperpolarized potentials, and impaired recovery from fast and slow inactivation. The LA-insensitive mutants N406K and F1760A exhibited reduced tonic and use-dependent block by methadone, and docking predictions positioned methadone in a cavity that was delimited by the residue F1760. Dextromethadone and levomethadone induced discrete stereo-selective effects on Na(v)1.5 channels.Methadone interacted with the LA-binding site to inhibit Na(v)1.5 channels. Our data suggest that these channels are a hitherto unrecognized molecular component contributing to cardiac arrhythmias induced by methadone.