Differences in local anaesthetic and antiepileptic binding in the inactivated state of human sodium channel Nav1.4.

Voltage-gated sodium channels play a vital role in nerve and muscle cells, enabling them to encode and transmit electrical signals. Currently, there exist several classes of drugs that aim to inhibit these channels for therapeutic purposes, including local anesthetics, antiepileptics and antiarrhythmics. However, sodium-channel-inhibiting drugs lack subtype specificity; instead, they inhibit all sodium channels in the human body. Improving understanding of the mechanisms of binding of existing nonselective drugs is important in providing insight into how subtype-selective drugs could be developed. This study used molecular dynamics simulations to investigate the binding of the antiepileptics carbamazepine and lamotrigine and the local anesthetic lidocaine in neutral and charged states to the recently resolved human Nav1.4 channel. Replica exchange solute tempering was used to enable greater sampling of each compound within the pore. It was found that all four compounds show similarities in their binding sites within the pore. However, the positions of the carbamazepine and lamotrigine did not occlude the center of the pore but preferentially bound to homologous domain DII and DIII. The charged and neutral forms of lidocaine positioned themselves more centrally in the pore, with more common interactions with DIV. The best localized binding site was for charged lidocaine, whose aromatic moiety interacted with Y1593, whereas the amine projected toward the selectivity filter. Comparisons with our previous simulations and published structures highlight potential differences between tonic and use-dependent block related to conformational changes occurring in the pore.

Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels.

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1 nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .

Cytotoxicity of amide-linked local anesthetics on melanoma cells via inhibition of Ras and RhoA signaling independent of sodium channel blockade.

BACKGROUND: Substantial clinical and preclinical evidence have indicated the association between amide-linked local anesthesia and the long-term outcomes of cancer patients. However, the potential effects of local anesthesia on cancer recurrence are inconclusive and the underlying mechanisms remain poorly understood. METHODS: We systematically examined the effects of three commonly used local anesthetics in melanoma cells and analyzed the underlying mechanisms focusing on small GTPases. RESULTS: Ropivacaine and lidocaine but not bupivacaine inhibited migration and proliferation, and induced apoptosis in melanoma cells. In addition, ropivacaine and lidocaine but not bupivacaine significantly augmented the in vitro efficacy of vemurafenib (a B-Raf inhibitor for melanoma with BRAF V600E mutation) and dacarbazine (a chemotherapeutic drug). Mechanistically, ropivacaine but not bupivacaine decreased the activities of Ras superfamily members with the dominant inhibitory effects on RhoA and Ras, independent of sodium channel blockade. Rescue studies using constitutively active Ras and Rho activator calpeptin demonstrated that ropivacaine inhibited migration mainly through RhoA whereas growth and survival were mainly inhibited through Ras in melanoma cells. We further detected a global reduction of downstream signaling of Ras and RhoA in ropivacaine-treated melanoma cells. CONCLUSION: Our study is the first to demonstrate the anti-melanoma activity of ropivacaine and lidocaine but not bupivacaine, via targeting small GTPases. Our findings provide preclinical evidence on how amide-linked local anesthetics could affect melanoma patients.

Natural Voltage-Gated Sodium Channel Ligands: Biosynthesis and Biology.

Natural product biosynthetic pathways are composed of enzymes that use powerful chemistry to assemble complex molecules. Small molecule neurotoxins are examples of natural products with intricate scaffolds which often have high affinities for their biological targets. The focus of this Minireview is small molecule neurotoxins targeting voltage-gated sodium channels (VGSCs) and the state of knowledge on their associated biosynthetic pathways. There are three small molecule neurotoxin receptor sites on VGSCs associated with three different classes of molecules: guanidinium toxins, alkaloid toxins, and ladder polyethers. Each of these types of toxins have unique structural features which are assembled by biosynthetic enzymes and the extent of information known about these enzymes varies among each class. The biosynthetic enzymes involved in the formation of these toxins have the potential to become useful tools in the efficient synthesis of VGSC probes.

Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model.

Experimental evidence regarding the risk of proarrhythmic potential of acehytisine is limited. We assessed its electropharmacological effect together with proarrhythmic potential at intravenous doses of 4 and 10 mg/kg (n = 6) using isoflurane-anesthetized guinea pigs in comparison with that of bepridil at 1 and 3 mg/kg, intravenously (n = 6). Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval, QTc, MAP90(sinus), MAP90(CL300) and MAP90(CL250). At supratherapeutic dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. Quantitative assessment showed that peak changes in P wave duration by acehytisine at 10 mg/kg were 1.7 times longer than bepridil, and in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than bepridil, respectively. Importantly, qualitative assessment indicated that bepridil increased beat-to-beat variability and J-Tpeakc in a dose-related manner, confirming a higher proarrhythmic risk, whereas such dose-related responses were not observed in acehytisine, suggesting a lower proarrhythmic risk. These results suggest that acehytisine exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity compared with bepridil, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.

The crystal structure of a voltage-gated sodium channel.

Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7 Šresolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ∼4.6 Šwide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism.

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

Understanding Sodium Channel Function and Modulation Using Atomistic Simulations of Bacterial Channel Structures.

Sodium channels are chief proteins involved in electrical signaling in the nervous system, enabling critical functions like heartbeat and brain activity. New high-resolution X-ray structures for bacterial sodium channels have created an opportunity to see how these proteins operate at the molecular level. An important challenge to overcome is establishing relationships between the structures and functions of mammalian and bacterial channels. Bacterial sodium channels are known to exhibit the main structural features of their mammalian counterparts, as well as several key functional characteristics, including selective ion conduction, voltage-dependent gating, pore-based inactivation and modulation by local anesthetic, antiarrhythmic and antiepileptic drugs. Simulations have begun to shed light on each of these features in the past few years. Despite deviations in selectivity signatures for bacterial and mammalian channels, simulations have uncovered the nature of the multiion conduction mechanism associated with Na(+) binding to a high-field strength site established by charged glutamate side chains. Simulations demonstrated a surprising level of flexibility of the protein, showing that these side chains are active participants in the permeation process. They have also uncovered changes in protein structure, leading to asymmetrical collapses of the activation gate that have been proposed to correspond to inactivated structures. These observations offer the potential to examine the mechanisms of state-dependent drug activity, focusing on pore-blocking and pore-based slow inactivation in bacterial channels, without the complexities of inactivation on multiple timescales seen in eukaryotic channels. Simulations have provided molecular views of the interactions of drugs, consistent with sites predicted in mammalian channels, as well as a wealth of other sites as potential new drug targets. In this chapter, we survey the new insights into sodium channel function that have emerged from studies of simpler bacterial channels, which provide an excellent learning platform, and promising avenues for mechanistic discovery and pharmacological development.

Convergent Evolution of Tetrodotoxin-Resistant Sodium Channels in Predators and Prey.

Convergent evolution of similar adaptive traits may arise from either common or disparate molecular and physiological mechanisms. The forces that determine the degree of underlying mechanistic similarities across convergent phenotypes are highly debated and poorly understood. Some garter snakes are able to consume newts that possess the channel blocking compound tetrodotoxin (TTX). Despite belonging to unrelated lineages, both the predators and prey have independently evolved remarkably similar physiological mechanisms of resistance to TTX that involve chemical and structural changes in voltage-gated sodium channels (NaV). The evolution of TTX resistance in this predator-prey pair constitutes a natural experiment that allows us to explore the causes of molecular convergence. Here, we review broad patterns of convergence at the level of amino acid changes in NaV channels of animals that evolved TTX resistance and make comparisons to known TTX-resistant channels that did not evolve under the selective pressures imposed by TTX. We conclude that convergence likely stems from the interplay of the target specificity of TTX and functional constraints of NaV that are shared among taxa. These and other factors can limit channel evolution to favor a few functionally permissible paths of adaptation, which can explain the observed predictability of changes to channel structure. By studying the functional causes of convergence in NaV channels, we can further our understanding of the role of these important channel proteins at the center of the evolution of the nervous system.

Parallel evolution of tetrodotoxin resistance in three voltage-gated sodium channel genes in the garter snake Thamnophis sirtalis.

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav's, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1-1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood-brain barrier. We also report the exon-intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

Intrinsic membrane plasticity via increased persistent sodium conductance of cholinergic neurons in the rat laterodorsal tegmental nucleus contributes to cocaine-induced addictive behavior.

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus implicated in reward processing and is one of the main sources of cholinergic afferents to the ventral tegmental area (VTA). Neuroplasticity in this structure may affect the excitability of VTA dopamine neurons and mesocorticolimbic circuitry. Here, we provide evidence that cocaine-induced intrinsic membrane plasticity in LDT cholinergic neurons is involved in addictive behaviors. After repeated experimenter-delivered cocaine exposure, ex vivo whole-cell recordings obtained from LDT cholinergic neurons revealed an induction of intrinsic membrane plasticity in regular- but not burst-type neurons, resulting in increased firing activity. Pharmacological examinations showed that increased riluzole-sensitive persistent sodium currents, but not changes in Ca(2+) -activated BK, SK or voltage-dependent A-type potassium conductance, mediated this plasticity. In addition, bilateral microinjection of riluzole into the LDT immediately before the test session in a cocaine-induced conditioned place preference (CPP) paradigm inhibited the expression of cocaine-induced CPP. These findings suggest that intrinsic membrane plasticity in LDT cholinergic neurons is causally involved in the development of cocaine-induced addictive behaviors.

The specificity of Av3 sea anemone toxin for arthropods is determined at linker DI/SS2-S6 in the pore module of target sodium channels.

Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site-3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3 was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determining Av3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp404 and His405 as a putative point of Av3 interaction with DmNav1. His405 conservation in arthropod Navs compared with tyrosine in vertebrate Navs may represent an ancient substitution that explains the contemporary selectivity of Av3. Trp404 and His405 localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.

Block of human cardiac sodium channels by lacosamide: evidence for slow drug binding along the activation pathway.

Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na(+) channels for its therapeutic action. Cardiac Na(+) channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na(+) channels. Lacosamide showed little effect on hNav1.5 Na(+) currents at 300 µM when cells were held at -140 mV. With 30-second conditioning pulses from -90 to -50 mV; however, hNav1.5 Na(+) channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70-80 µM. Higher IC50 values were found at -110 and -30 mV. The development of lacosamide block at -70 mV was slow in wild-type Na(+) channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at -70 mV for 10 seconds was relatively rapid in wild-type Na(+) channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na(+) currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na(+) channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na(+) channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway.

Activation of the prelimbic medial prefrontal cortex induces anxiety-like behaviors via N-Methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in mice.

We investigated the possible roles of the prelimbic medial prefrontal cortex (PL) in the regulation of anxiety-like behaviors by pharmacologically activating the terminals of neuronal inputs or postsynaptic efferent neurons with a sodium channel activator veratrine. The extracellular glutamate levels were measured by in vivo microdialysis, and the behaviors were assessed with the open field (OF) test in mice simultaneously. The samples were collected every 10 min for 60 min, as basal levels of glutamate. The medium containing drugs were perfused for 30 min. The OF test was performed in the last 10 min of drug perfusion. After the drug treatments, the perfusion medium containing drugs was switched back to perfusion medium without drugs, and then samples were collected for another 90 min. The extracellular glutamate levels were significantly elevated after local perfusion of veratrine in the PL. At the same time, perfusion of veratrine in the PL produced anxiety-like behaviors in mice. Local coperfusion of a sodium channel blocker, lamotrigine, completely diminished the veratrine-induced elevated extracellular glutamate levels and the behavioral changes. Local coperfusion of an NMDA receptor antagonist, MK-801, but not a non-NMDA (AMPA/kainate) receptor antagonist, CNQX, completely diminished the behavioral changes without any effects on the veratrine-induced elevated extracellular glutamate levels. This study demonstrates that the activation of the PL with veratrine induces anxiety-like behaviors via NMDA receptor-mediated glutamatergic neurotransmission in mice.

AdE-1, a new inotropic Na(+) channel toxin from Aiptasia diaphana, is similar to, yet distinct from, known anemone Na(+) channel toxins.

Heart failure is one of the most prevalent causes of death in the western world. Sea anemone contains a myriad of short peptide neurotoxins affecting many pharmacological targets, several of which possess cardiotonic activity. In the present study we describe the isolation and characterization of AdE-1 (ion channel modifier), a novel cardiotonic peptide from the sea anemone Aiptasia diaphana, which differs from other cnidarian toxins. Although AdE-1 has the same cysteine residue arrangement as sea anemone type 1 and 2 Na(+) channel toxins, its sequence contains many substitutions in conserved and essential sites and its overall homology to other toxins identified to date is low (<36%). Physiologically, AdE-1 increases the amplitude of cardiomyocyte contraction and slows the late phase of the twitch relaxation velocity with no induction of spontaneous twitching. It increases action potential duration of cardiomyocytes with no effect on its threshold and on the cell's resting potential. Similar to other sea anemone Na(+) channel toxins such as Av2 (Anemonia viridis toxin II), AdE-1 markedly inhibits Na(+) current inactivation with no significant effect on current activation, suggesting a similar mechanism of action. However, its effects on twitch relaxation velocity, action potential amplitude and on the time to peak suggest that this novel toxin affects cardiomyocyte function via a more complex mechanism. Additionally, Av2's characteristic delayed and early after-depolarizations were not observed. Despite its structural differences, AdE-1 physiologic effectiveness is comparable with Av2 with a similar ED(50) value to blowfly larvae. This finding raises questions regarding the extent of the universality of structure-function in sea anemone Na(+) channel toxins.

µ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve.

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na(V)1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na(V)1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 µ-conopeptides for their ability to block rodent Na(V)1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na(V)1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A- and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A- and C-fibers were Na(V)1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na(V)1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na(V)1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of µ-conopeptides should be useful in identifying the functional contributions of Na(V)1 isoforms in other preparations.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.

Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers.

Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and thereby the excitability of all neurons, not just sensory neurons. We tested the possibility of selectively blocking the excitability of primary sensory nociceptor (pain-sensing) neurons by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel. Here we show that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability. Inhibition by co-applied QX-314 and capsaicin was restricted to neurons expressing TRPV1. Injection of QX-314 together with capsaicin into rat hindpaws produced a long-lasting (more than 2 h) increase in mechanical and thermal nociceptive thresholds. Long-lasting decreases in pain sensitivity were also seen with regional injection of QX-314 and capsaicin near the sciatic nerve; however, in contrast to the effect of lidocaine, the application of QX-314 and capsaicin together was not accompanied by motor or tactile deficits.

Binding modes of µ-conotoxin to the bacterial sodium channel (NaVAb).

Polypeptide toxins isolated from the venom of cone snails, known as µ-conotoxins, block voltage-gated sodium channels by physically occluding the ion-conducting pathway. Using molecular dynamics, we show that one subtype of µ-conotoxins, PIIIA, effectively blocks the bacterial voltage-gated sodium channel Na(V)Ab, whose crystal structure has recently been elucidated. The spherically shaped toxin, carrying a net charge of +6 e with six basic residues protruding from its surface, is attracted by the negatively charged residues on the vestibular wall and the selectivity filter of the channel. The side chain of each of these six arginine and lysine residues can wedge into the selectivity filter, whereas the side chains of other basic residues form electrostatic complexes with two acidic residues on the channel. We construct the profile of potential of mean force for the unbinding of PIIIA from the channel, and predict that PIIIA blocks the bacterial sodium channel with subnanomolar affinity.