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BACKGROUND: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. METHODS: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. RESULTS: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular

CA1 pyramidal neurons undergo intense morphological and electrophysiological changes from the second to third postnatal weeks in rats throughout a critical period associated with the emergence of exploratory behavior. Using whole cell current-clamp recordings in vitro and neurochemical methods, we studied the development of the somatic action potential (AP) waveform and some of the underlying channels in this critical period. At the third postnatal week, APs showed a more hyperpolarized threshold, higher duration and amplitude. Subthreshold depolarization broadened APs and depolarized their peak overshoots more pronouncedly in immature neurons (2 weeks old). These features were mimicked by pharmacologically blocking the fast-inactivating A-type potassium current (IA) and matched well with the higher concentrations of Kv4.2 and Kv4.3 and the lower concentrations of BK and Kv1.2 channels detected by Western blotting. Repetitive stimulation with high frequency trains (50 Hz) reproduced AP broadening associated to inactivation of the A-type current in immature cells. Moreover, repetitive firing showed changes in AP amplitude consistent with the inactivation of both sodium and potassium subthreshold currents, which resulted in higher AP amplitudes in the more immature neurons. We propose that maturation of AP waveform and excitability in this critical developmental period could be related to the onset of exploratory behaviors., Ministerio de Ciencia e Innovación, Depto. de Fisiología, Fac. de Medicina, TRUE, pub

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Ever since Hodgkin and Huxley described the sodium dependence in action potential nearly 70 years ago, neuroscientists have discovered an entire family of voltage-gated sodium channels (NaVs) in neuronal cells. Uncovering the differential distributions of these NaV isoforms between and within each type of neuron helped us understand the distinct roles they play in cellular functions. So crucial to normal neuronal physiology, genetic variants that result in protein structure changes can cause pronounced alteration in brain function and lead to disease. NaV1.2, encoded by SCN2A, is one NaV isoform whose genetic variants have been linked to neurodevelopmental disorders such as autism spectrum disorder (ASD). Studies have uncovered the importance of NaV1.2 in neuronal intrinsic properties and synaptic physiology in the forebrain. However, how these channels maintain normal cellular functions in the cerebellum, in which some of the strongest expression of NaV1.2 are detected, is unclear. The cerebellum is heavily implicated in ASD due to its functions in motor learning, fine movement control, and more recently discovered, cognition. Therefore, understanding how SCN2A dysfunctions alter cerebellar circuit has become critical to study disease manifestation in channelopathies. In my thesis work, I found that Scn2a haploinsufficiency in mice decreased cerebellar granule cell excitability, impaired high-frequency action potential propagation along their axons, and ultimately compromised the downstream synaptic plasticity that is critical to cerebellar learning. I also used cerebellum-dependent vestibulo-ocular reflex (VOR) to uncover that mice with Scn2a heterozygosity exhibited saturated VOR gain and deficit in VOR adaptation, which are correlated with abnormal Purkinje cell firing pattern during behavior. In addition, children with LoF SCN2A variants showed a similarly elevated baseline VOR gain. Lastly, I revealed that the VOR adaptation deficit in mice can be rescued by u

In this thesis we asked the questions how ion signaling and the periodic organization at the AIS shape the generation of action potentials. In order to answer these questions, we need to bridge functional investigations with high resolution structural reconstructions. In Chapter 2, we used optical calcium recordings and identified three calcium entry pathways in the AIS. As expected, calcium is released from internal stores and enters through calcium channels, however, surprisingly, we also observed calcium entering through sodium channels. We estimated that the conductivity ratio of sodium channels for calcium is small, but because they are present at a high density at the AIS, they do form a major and rapid source of calcium. In Chapter 3, we investigated whether the calcium-dependent BK channel was a downstream target for calcium in the AIS. We implemented a novel technique to use light patterning of a fluorescent voltage reporter to obtain highly accurate measures of the action potential shape in the axon. BK channels were indeed activated during the action potential at the AIS, forming a link between calcium entry and action potential repolarization. Together, the complex of calcium and BK channels mediated high-frequency burst firing, an important feature of the cell type that we studied. In Chapter 4, we developed a novel optical method to perform high resolution microscopy deep inside tissue, where the neurons are in an intact three-dimensional context. Because biological tissue is not transparent, light traveling through tissue suffers from distortions, which makes microscopy at depth problematic. To overcome this obstacle, we used a deformable mirror to counteract the light distortions and enable high resolution microscopy inside biological tissue. We used this method to perform both live experiments and high-resolution microscopy from the same neuron, demonstrating that this method can bridge the structure-function relationship in neurons. Together, the e

Chronic pain affects nearly 1 in 4 individuals worldwide. Despite its global spread, patients systematically report lack of efficacy and severe side effects from available treatments, leading to years of trial-and-error management for little to no improvement. Lacosamide, originally developed for epilepsy, was found to reduce pain in a sub-group of patients. This research shows that some mutations in genes involved with the transmission of pain signals can alter drug effects. Another discovery was that one particular mutation, which completely prevented pain relief in patients, was responsible for linking the lacosamide molecules to their receptors, which then block the pain signal. The identification of such mutations can predict how a patient will respond to the drug before even receiving it. Nowadays, the patient’s own cells can be tested against different drugs. Stem cells can be obtained from a skin sample and turned into neurons. However, they are imperfect copies of the patient’s actual neurons. This thesis shows ways to improve cell quality, notably by injecting them into the rat nervous system and by using electrical stimulation.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

It has been demonstrated experimentally that slow and fast conduction waves with distinct conduction velocities can occur in the same nerve system depending on the strength or the form of the stimulus, which give rise to two modes of nerve functions. However, the mechanisms remain to be elucidated. In this study, we use computer simulations of the cable equation with modified Hodgkin-Huxley kinetics and analytical solutions of a simplified model to show that stimulus-dependent slow and fast waves recapitulating the experimental observations can occur in the cable, which are the two stable conduction states of a bistable conduction behavior. The bistable conduction is caused by a positive feedback loop of the wavefront upstroke speed, mediated by the sodium channel inactivation properties. Although the occurrence of bistable conduction only requires the presence of the sodium current, adding a calcium current to the model further promotes bistable conduction by potentiating the slow wave. We also show that the bistable conduction is robust, occurring for sodium and calcium activation thresholds well within the experimentally determined ones of the known sodium and calcium channel families. Since bistable conduction can occur in the cable equation of Hodgkin-Huxley kinetics with a single inward current, i.e., the sodium current, it can be a generic mechanism applicable to stimulus-dependent fast and slow conduction not only in the nerve systems but also in other electrically excitable systems, such as cardiac muscles.

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale

Emerging marine biotoxins such as ciguatoxins and pyrethroid compounds, widely used in agriculture, are independently treated as environmental toxicants. Their maximum residue levels in food components are set without considering their possible synergistic effects as consequence of their interaction with the same cellular target. There is an absolute lack of data on the possible combined cellular effects that biological and chemical pollutants, may have. Nowadays, an increasing presence of ciguatoxins in European Coasts has been reported and these toxins can affect human health. Similarly, the increasing use of phytosanitary products for control of food plagues has raised exponentially during the last decades due to climate change. The lack of data and regulation evaluating the combined effect of environmental pollutants with the same molecular target led us to analyse their in vitro effects. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a profound synergistic effect in voltage-dependent sodium channels. These food pollutants act by decreasing the maximum peak inward sodium currents and hyperpolarizing the sodium channels activation, effects that are boosted by the simultaneous presence of both compounds. A fact that highlights the need to re-evaluate their limits in feedstock as well as their potential in vivo toxicity considering that they act on the same cellular target. Moreover, this work sets the cellular basis to further apply this type of studies to other water and food pollutants that may act synergistically and thus implement the corresponding regulatory limits taking into account its presence in a healthy diet

The olfactory system of the fruit fly Drosophila melanogaster is an invaluable model for understanding circuit function. Composed mainly of olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs), it is an analogous structure to mammalian olfactory systems. Of these cell types, LNs are particularly intriguing. These neurons are found in a variety of morphologies and with differing neurotransmitter and receptor profiles. Given their heterogeneity, it is critical to gain an understanding of their roles in olfactory circuits. In this work, I probe the physiology and functions of two unique subpopulations of LNs in the antennal lobe (AL). In the first population, I demonstrate LNs which respond to extrasynaptic, paracrine levels of serotonergic modulation. These LNs then engage in postsynaptic inhibition and subtractive gain control, which is contrary to typical LNs. The second population I characterize are previously undescribed nonspiking LNs in the fly AL. Nonspiking cells are common to insect olfaction as well as other sensory pathways in vertebrates. I find that these neurons are likely to be electrotonically compartmentalized, such that activation within individual regions does not propagate across the whole cell, suggesting roles in previously unexplained mechanisms such as intraglomerular inhibition. The results of this work suggest more heterogeneity in Drosophila LNs than previously assumed and cements the importance of interneuron contribution to neuronal function.

CRediT authorship contribution statement Marina Arribas-Blázquez: performed research. Dolores Piniella: performed research. Luis A. Olivos-Oré: analyzed data and wrote the paper, performed research. David Bartolomé-Martín: performed research. Cristiana Leite: performed research. Cecilio Giménez: designed research. Antonio R. Artalejo: designed research, analyzed data and wrote the paper. Francisco Zafra: designed research, analyzed data and wrote the paper., The voltage-sensitive sodium channel NaV1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate NaV1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of NaV1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na+ currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing NaV1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous NaV1.1 in primary cultures were identified by expressing a fluorescent protein under the NaV1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis., Fundación Ramón Areces, MINECO (RTI2018-098712-B-I00), Fundación Síndrome de Dravet, Sección Deptal. de Farmacología y Toxicología (Veterinaria), Fac. de Veterinaria, TRUE, pub

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (NaV) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on NaV function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a NaV DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog NaVs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog NaVs. We also show that captivity-raised poison frogs are resistant to two NaV-directed toxins, BTX and saxitoxin (STX), even though they bear NaVs sensitive to both. Moreover, we demonstrate that the amphibian STX "toxin sponge" protein saxiphilin is able to protect and rescue NaVs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.

It is imperative to develop better approaches to predict how antiarrhythmic drugs with multiple interactions and targets may alter the overall electrical and/or mechanical function of the heart. Safety Pharmacology studies have provided new insights into the multi-target effects of many different classes of drugs and have been aided by the addition of robust new in vitro and in silico technology. The primary focus of Safety Pharmacology studies has been to determine the risk profile of drugs and drug candidates by assessing their effects on repolarization of the cardiac action potential. However, for decades experimental and clinical studies have described substantial and potentially detrimental effects of Na+ channel blockers in addition to their well-known conduction slowing effects. One such side effect, associated with administration of some Na+ channel blocking drugs is negative inotropy. This reduces the pumping function of the heart, thereby resulting in hypotension. Flecainide is a well-known example of a Na+ channel blocking drug, that exhibits strong rate-dependent block of INa and may cause negative cardiac inotropy. While the phenomenon of Na+ channel suppression and resulting negative inotropy is well described, the mechanism(s) underlying this effect are not. Here, we set out to use a modeling and simulation approach to reveal plausible mechanisms that could explain the negative inotropic effect of flecainide. We utilized the Grandi-Bers model [1] of the cardiac ventricular myocyte because of its robust descriptions of ion homeostasis in order to characterize and resolve the relative effects of QRS widening, flecainide off-target effects and changes in intracellular Ca2+ and Na+ homeostasis. The results of our investigations and predictions reconcile multiple data sets and illustrate how multiple mechanisms may play a contributing role in the flecainide induced negative cardiac inotropic effect.

The voltage-sensitive sodium channel Na1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate Na1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of Na1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing Na1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous Na1.1 in primary cultures were identified by expressing a fluorescent protein under the Na1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis.

Dysfunction in the gene SCN2A has a strong association with autism spectrum disorder (ASD). SCN2A encodes the protein NaV1.2, a voltage-gated sodium channel important for the initiation and propagation of action potential in excitatory neurons in the developing cortex. ASD-associated SCN2A variants impair the function of NaV1.2, however, the resulting consequences of NaV1.2 dysfunction for ASD are unknown. In this dissertation, I show that NaV1.2 is unexpectedly critical for dendritic excitability and synaptic function in mature pyramidal neurons, in addition to regulating axonal excitability early in development. Scn2a-haploinsufficiency disrupts action potential propagation into the dendritic arbor, impairing synaptic plasticity and function, even when NaV1.2 expression is disrupted in a cell-autonomous fashion late in development. Homozygous Scn2a-loss further exacerbates dendritic excitability deficits, yet paradoxically increases axonal excitability, likely resulting from compensation by other sodium channel genes. Encouragingly, restoring Scn2a expression in mature neurons appears sufficient to rescue dendritic and synaptic impairments. CRISPR activators are demonstrated as a variable approach for exogenously increasing Scn2a expression, restoring dendritic excitability and synaptic function in Scn2a-haploinsufficient mice. Overall, these results reveal a novel dendritic function for NaV1.2, provide insight into cellular mechanisms underlying circuit and behavioral dysfunction in ASD, and identify potential therapeutic avenues for loss-of-function SCN2A cases and other haploinsufficient ASD genes.

Malfunctions of voltage-gated sodium and calcium channels (encoded by SCNxA and CACNA1x family genes, respectively) have been associated with severe neurologic, psychiatric, cardiac, and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) that often corresponds not only to clinical disease manifestations but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. On the basis of known gene-disease mechanisms of 19 different diseases, we inferred LOF (n = 518) and GOF (n = 309) likely pathogenic variants from the disease phenotypes of variant carriers. By training a machine learning model on sequence- and structure-based features, we predicted LOF or GOF effects [area under the receiver operating characteristics curve (ROC) = 0.85] of likely pathogenic missense variants. Our LOF versus GOF prediction corresponded to molecular LOF versus GOF effects for 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and was validated in exome-wide data from 21,703 cases and 128,957 controls. We showed respective regional clustering of inferred LOF and GOF nucleotide variants across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCNxA/CACNA1x family genes.

BackgroundRapid delayed rectifier K+ current (IKr) and late Na+ current (INaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death.MethodsPhysiological self AP-clamp was used to measure INaL and IKr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between IKr and INaL affects repolarization stability in health and disease conditions.ResultsWe found comparable amount of net charge carried by IKr and INaL during the physiological AP, suggesting that outward K+ current via IKr and inward Na+ current via INaL are in balance during physiological repolarization. Remarkably, IKr and INaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block IKr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na+ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit INaL sufficiently restored APD to control in both cases. Importantly, INaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, INaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by INaL inhibition, increased by IKr inhibition, and restored by combined INaL and IKr inhibitions.ConclusionsOur data demonstrate that IKr and INaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic curre

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation

BackgroundDiabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus.Methods and ResultsMale plasminogen knockout (plasminogen-deficient [Plg(-/-)]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg(-/-) mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg(-/-) control mice, ANGII did not change blood pressure in diabetic Plg(-/-) mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg(-/-) mice with or without diabetes mellitus. Full-length gamma-ENaC and alpha-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa gamma-ENaC cleavage product was increased in diabetic versus nondiabetic mice.ConclusionsPlasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.

A series of amino-2-cyclohexyl ester derivatives were studied for their ion channel blocking and antiarrhythmic actions in the rat and a structure-activity analysis was conducted. The compounds are similar in chemical structure except for ionizable amine groups (pK values 6.1-8.9) and the positional arrangements of aromatic naphthyl moieties. Ventricular arrhythmias were produced in rats by coronary-artery occlusion or electrical stimulation. The electrophysiological effects of these compounds on rat heart sodium channels (Nav1.5) expressed in Xenopus laevis oocytes and transient outward potassium currents (Kv4.3) from isolated rat ventricular myocytes were examined. The compounds reduced the incidence of ischemia-related arrhythmias and increased current threshold for induction of ventricular fibrillo-flutter (VFt) dose-dependently. As pK increased compounds showed a diminished effectiveness against ischemia-induced arrhythmias, and were less selective for ischemia- versus electrically-induced arrhythmias. Where tested, compounds produced a concentration-dependent tonic block of Nav1.5 channels. An increased potency for inhibition of Nav1.5 occurred when the external pH (pHo) was reduced to 6.5. Some compounds inhibited Kv4.3 in a pH-independent manner. Overall, the differences in antiarrhythmic and ion channel blocking properties in this series of compounds can be explained by differences in chemical structure. Antiarrhythmic activity for the amino-2-cyclohexyl ester derivatives is likely a function of mixed ion channel blockade in ischemic myocardium. These studies show that drug inhibition of Nav1.5 occurred at lower concentrations than Kv4.3 and was more sensitive to changes in the ionizable amine groups rather than on positional arrangements of the naphthyl constituents. These results offer insight into antiarrhythmic mechanisms of drug-ion channel interactions.

The human voltage-gated sodium channel, hNaV1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNaV1.5-targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNaV1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNaV1.5 and will be useful for rational design of novel therapeutics.

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.

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). Here, we find that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket 25 (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK25 mediates Ca2+-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK25 expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance our understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species.

Potassium (K+) secretion by kidney tubule cells is central to electrolyte homeostasis in mammals. In the K+ secretory "principal" cells of the distal nephron, electrogenic Na+ transport by the epithelial sodium channel (ENaC) generates the electrical driving force for K+ transport across the apical membrane. Regulation of this process is attributable in part to aldosterone, which stimulates the gene transcription of the ENaC-regulatory kinase, SGK1. However, a wide range of evidence supports the conclusion that an unidentified aldosterone-independent pathway exists. We show here that in principal cells, K+ itself acts through the type 2 mTOR complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion. The effect depends on changes in K+ concentration on the blood side of the cells, and requires basolateral membrane K+-channel activity. However, it does not depend on changes in aldosterone, or on enhanced distal delivery of Na+ from upstream nephron segments. These data strongly support the idea that K+ is sensed directly by principal cells to stimulate its own secretion by activating the mTORC2-SGK1 signaling module, and stimulate ENaC. We propose that this local effect acts in concert with aldosterone and increased Na+ delivery from upstream nephron segments to sustain K+ homeostasis.

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.

Excessive alcohol use is extremely prevalent in the United States, particularly among trauma-exposed individuals. While several studies have examined genetic influences on alcohol use and related problems, this has not been studied in the context of trauma-exposed populations. We report results from a genome-wide association study of alcohol consumption and associated problems as measured by the alcohol use disorders identification test (AUDIT) in a trauma-exposed cohort. Results indicate a genome-wide significant association between total AUDIT score and rs1433375 [N = 1036, P = 2.61 × 10-8 (dominant model), P = 7.76 × 10-8 (additive model)], an intergenic single-nucleotide polymorphism located 323 kb upstream of the sodium channel and clathrin linker 1 (SCLT1) at 4q28. rs1433375 was also significant in a meta-analysis of two similar, but independent, cohorts (N = 1394, P = 0.0004), the Marine Resiliency Study and Systems Biology PTSD Biomarkers Consortium. Functional analysis indicated that rs1433375 was associated with SCLT1 gene expression and cortical-cerebellar functional connectivity measured via resting state functional magnetic resonance imaging. Together, findings suggest a role for sodium channel regulation and cerebellar functioning in alcohol use behavior. Identifying mechanisms underlying risk for problematic alcohol use in trauma-exposed populations is critical for future treatment and prevention efforts.

BackgroundRSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors.MethodsThe pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity.ResultsIn rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized.ConclusionsRSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

BACKGROUND: RSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors. METHODS: The pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity. RESULTS: In rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized. CONCLUSIONS: RSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Channelopathies are disorders caused by abnormal ion channel function in differentiated excitable tissues. We discovered a unique neurodevelopmental channelopathy resulting from pathogenic variants in SCN3A, a gene encoding the voltage-gated sodium channel NaV1.3. Pathogenic NaV1.3 channels showed altered biophysical properties including increased persistent current. Remarkably, affected individuals showed disrupted folding (polymicrogyria) of the perisylvian cortex of the brain but did not typically exhibit epilepsy; they presented with prominent speech and oral motor dysfunction, implicating SCN3A in prenatal development of human cortical language areas. The development of this disorder parallels SCN3A expression, which we observed to be highest early in fetal cortical development in progenitor cells of the outer subventricular zone and cortical plate neurons and decreased postnatally, when SCN1A (NaV1.1) expression increased. Disrupted cerebral cortical folding and neuronal migration were recapitulated in ferrets expressing the mutant channel, underscoring the unexpected role of SCN3A in progenitor cells and migrating neurons.

Loss-of-function mutations in SCN1A cause Dravet syndrome (DS), a catastrophic childhood epilepsy in which patients experience comorbid behavioral conditions, including movement disorders, sleep abnormalities, anxiety, and intellectual disability. To study the functional consequences of voltage-gated sodium channel mutations, we use zebrafish with a loss-of-function mutation in scn1lab, a zebrafish homolog of human SCN1A. Homozygous scn1labs552/s552 mutants exhibit early-life seizures, metabolic deficits, and early death. Here, we developed in vivo assays using scn1labs552 mutants between 3 and 6 d postfertilization (dpf). To evaluate sleep disturbances, we monitored larvae for 24 h with locomotion tracking software. Locomotor activity during dark (night phase) was significantly higher in mutants than in controls. Among anticonvulsant drugs, clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved at night for scn1labs552 mutant larvae. To monitor exploratory behavior in an open field, we tracked larvae in a novel arena. Mutant larvae exhibited impaired exploratory behavior, with increased time spent near the edge of the arena and decreased mobility, suggesting greater anxiety. Both clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved and increased time spent in the center of the arena. Counting inhibitory neurons in vivo revealed no differences between scn1labs552 mutants and siblings. Taken together, our results demonstrate conserved features of sleep, anxiety, and movement disorders in scn1lab mutant zebrafish, and provide evidence that a zebrafish model allows effective tests of treatments for behavioral comorbidities associated with DS.

The homeostatic control of presynaptic neurotransmitter release stabilizes information transfer at synaptic connections in the nervous system of organisms ranging from insect to human. Presynaptic homeostatic signaling centers upon the regulated membrane insertion of an amiloride-sensitive degenerin/epithelial sodium (Deg/ENaC) channel. Elucidating the subunit composition of this channel is an essential step toward defining the underlying mechanisms of presynaptic homeostatic plasticity (PHP). Here, we demonstrate that the ppk1 gene encodes an essential subunit of this Deg/ENaC channel, functioning in motoneurons for the rapid induction and maintenance of PHP. We provide genetic and biochemical evidence that PPK1 functions together with PPK11 and PPK16 as a presynaptic, hetero-trimeric Deg/ENaC channel. Finally, we highlight tight control of Deg/ENaC channel expression and activity, showing increased PPK1 protein expression during PHP and evidence for signaling mechanisms that fine tune the level of Deg/ENaC activity during PHP.

Amino acid taste is expected to be a universal property among animals. Although sweet, bitter, salt, and water tastes have been well characterized in insects, the mechanisms underlying amino acid taste remain elusive. From a Drosophila RNAi screen, we identify an ionotropic receptor, Ir76b, as necessary for yeast preference. Using calcium imaging, we identify Ir76b+ amino acid taste neurons in legs, overlapping partially with sweet neurons but not those that sense other tastants. Ir76b mutants have reduced responses to amino acids, which are rescued by transgenic expression of Ir76b and a mosquito ortholog AgIr76b. Co-expression of Ir20a with Ir76b is sufficient for conferring amino acid responses in sweet-taste neurons. Notably, Ir20a also serves to block salt response of Ir76b. Our study establishes the role of a highly conserved receptor in amino acid taste and suggests a mechanism for mutually exclusive roles of Ir76b in salt- and amino-acid-sensing neurons.

Pathogens use a variety of secreted and surface proteins to interact with and manipulate their hosts, but a systematic approach for identifying such proteins has been lacking. To identify these host-exposed proteins, we used spatially restricted enzymatic tagging followed by mass spectrometry analysis of Caenorhabditis elegans infected with two species of Nematocida microsporidia. We identified 82 microsporidia proteins inside of intestinal cells, including several pathogen proteins in the nucleus. These microsporidia proteins are enriched in targeting signals, are rapidly evolving and belong to large Nematocida-specific gene families. We also find that large, species-specific families are common throughout microsporidia species. Our data suggest that the use of a large number of rapidly evolving species-specific proteins represents a common strategy for microsporidia to interact with their hosts. The unbiased method described here for identifying potential pathogen effectors represents a powerful approach to study a broad range of pathogens.

A majority of studies on the brain are focused at either the molecular level or organ level. Rarely do we see models that attempt to relate neuronal behaviors to symptoms of traumatic brain injury. Well-documented studies of the electrophysiology of the neuron provide the opportunity to examine the effects of traumatic brain injury via the integration of computational mechanics and mathematical models of the neuron. This work focuses on the consequences of imposing mechanical insults to neurons.

The mammalian superior colliculus (SC) is a midbrain structure that integrates multimodal sensory inputs and computes commands to initiate rapid eye movements. SC neurons burst with the sudden onset of a visual stimulus, followed by persistent activity that may underlie shifts of attention and decision making. Experiments in vitro suggest that circuit reverberations play a role in the burst activity in the SC, but the origin of persistent activity is unclear. In the present study we characterized an afterdepolarization (ADP) that follows action potentials in slices of rat SC. Population responses seen with voltage-sensitive dye imaging consisted of rapid spikes followed immediately by a second distinct depolarization of lower amplitude and longer duration. Patch-clamp recordings showed qualitatively similar behavior: in nearly all neurons throughout the SC, rapid spikes were followed by an ADP. Ionic and pharmacological manipulations along with experiments with current and voltage steps indicated that the ADP of SC neurons arises from Na(+) current that either persists or resurges following Na(+) channel inactivation at the end of an action potential. Comparisons of pharmacological properties and frequency dependence revealed a clear parallel between patch-clamp recordings and voltage imaging experiments, indicating a common underlying membrane mechanism for the ADP in both single neurons and populations. The ADP can initiate repetitive spiking at intervals consistent with the frequency of persistent activity in the SC. These results indicate that SC neurons have intrinsic membrane properties that can contribute to electrical activity that underlies shifts of attention and decision making.

BackgroundThe QT interval is a phase of the cardiac cycle that corresponds to action potential duration (APD) including cellular repolarization (T-wave). In both clinical and experimental settings, prolongation of the QT interval of the electrocardiogram (ECG) and related proarrhythmia have been so strongly associated that a prolonged QT interval is largely accepted as surrogate marker for proarrhythmia. Accordingly, drugs that prolong the QT interval are not considered for further preclinical development resulting in removal of many promising drugs from development. While reduction of drug interactions with hERG is an important goal, there are promising means to mitigate hERG block. Here, we examine one possibility and test the hypothesis that selective inhibition of the cardiac late Na current (INaL) by the novel compound GS-458967 can suppress proarrhythmic markers.Methods and resultsNew experimental data has been used to calibrate INaL in the Soltis-Saucerman computationally based model of the rabbit ventricular action potential to study effects of GS-458967 on INaL during the rabbit ventricular AP. We have also carried out systematic in silico tests to determine if targeted block of INaL would suppress proarrhythmia markers in ventricular myocytes described by TRIaD: Triangulation, Reverse use dependence, beat-to-beat Instability of action potential duration, and temporal and spatial action potential duration Dispersion.ConclusionsOur computer modeling approach based on experimental data, yields results that suggest that selective inhibition of INaL modifies all TRIaD related parameters arising from acquired Long-QT Syndrome, and thereby reduced arrhythmia risk. This study reveals the potential for adjunctive pharmacotherapy via targeted block of INaL to mitigate proarrhythmia risk for drugs with significant but unintended off-target hERG blocking effects.

In a recent edition of Biochemical Journal, Mistry et al. described the discovery of a novel protein complex, formed from the epithelial sodium channel (ENaC) and the sodium chloride cotransporter (NCC) [Mistry et al. (2016) Biochem. J. 473, 3237–3252]. The importance of these two proteins in the regulation of salt balance and blood pressure has long been known, as has their overlapping expression in the distal convoluted tubule of the kidney. The new study by Mistry et al. now demonstrates their physical interaction in the kidney and when heterologously co-expressed. Furthermore, the authors demonstrate some degree of functional co-dependence between ENaC and NCC, with pharmacological inhibition of the latter diminishing activity of the former when the two are co-assembled. This novel and potentially important interaction adds to a growing number of recently identified channel-transporter ('chansporter') complexes, which together constitute an emerging theme in cell signaling.

In a recent edition of Biochemical Journal, Mistry et al. described the discovery of a novel protein complex, formed from the epithelial sodium channel (ENaC) and the sodium chloride cotransporter (NCC) [Mistry et al. (2016) Biochem. J. 473, 3237–3252]. The importance of these two proteins in the regulation of salt balance and blood pressure has long been known, as has their overlapping expression in the distal convoluted tubule of the kidney. The new study by Mistry et al. now demonstrates their physical interaction in the kidney and when heterologously co-expressed. Furthermore, the authors demonstrate some degree of functional co-dependence between ENaC and NCC, with pharmacological inhibition of the latter diminishing activity of the former when the two are co-assembled. This novel and potentially important interaction adds to a growing number of recently identified channel-transporter (chansporter) complexes, which together constitute an emerging theme in cell signaling.

It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated “ephaptic” mechanisms, facilitating propagation of action potentials in tandem with direct GJmediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus andwas associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.

This paper reviews Drosophila voltage-gated Na(+) channel mutations encoded by the para (paralytic) gene and their contributions to seizure disorders in the fly. Numerous mutations cause seizure-sensitivity, for example, para(bss1), with phenotypes that resemble human intractable epilepsy in some aspects. Seizure phenotypes are also seen with human GEFS+ spectrum mutations that have been knocked into the Drosophila para gene, para(GEFS+) and para(DS) alleles. Other para mutations, para(ST76) and para(JS) act as seizure-suppressor mutations reverting seizure phenotypes in other mutants. Seizure-like phenotypes are observed from mutations and other conditions that cause a persistent Na(+) current through either changes in mRNA splicing or protein structure.

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.

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information.