Sodium Channel Availability Research Articles

Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

30ml 0.5% isobaric levobupivacaine over 30ml 0.5% bupivacaine used for interscalene brachial plexus block: Haemodynamic parameters

The primary cardiac electrophysiologic effect of local anaesthetic is a decrease in the maximum rate of depolarization in the purkinje fibres and ventricular muscle. This is due to a decrease in the availability of sodium channels. Action potential duration and the effective refractory period is alsodecreased. Sixty patients aged between 18yrs and 60yrs of physical status ASA grade 1 and ASA grade 2 undergoing elective upper limb surgeries were included in the study after ethical clearance from the college ethical committee. Each patient was visited pre-operatively and the procedure explained and written informed consent was obtained. The systolic blood pressure measurement done at various time intervals did not show any statistically significant difference (p>0.05) between the two groups. The diastolic blood pressure measurement done at various time intervals show no statistical difference between the two groups (p>0.05).

Membrin/GOSR2 is a novel NaV1.5-interacting protein modulating cardiac conduction

Abstract Background Genome-wide association studies have associated a locus spanning GOSR2 with QRS- and QT-interval. GOSR2 encodes Membrin, a protein located at the cis-Golgi, which plays a role in protein trafficking. Altered trafficking of the cardiac sodium channel (NaV1.5), encoded by SCN5A, has been shown to reduce cardiac conduction. Purpose To explore the modulatory role of Membrin on cardiac conduction and sodium channel availability. Methods and results Tandem Affinity Purification in H10 cells (derived from neonatal rat cardiomyocytes) overexpressing the NaV1.5 C-terminus identified Membrin as a putative interactor of NaV1.5. We subsequently confirmed the interaction between NaV1.5 and Membrin by means of a co-immunoprecipitation assay in HEK293A cells that overexpress NaV1.5 and Membrin. To investigate whether Membrin affects cardiac conduction we recorded optical action potentials from the left ventricle (LV) of Langendorff-perfused hearts from Gosr2+/− mice and wild type (WT) littermate controls. Conduction velocity was measured at steady state pacing (cycle length 120ms) and at the minimal possible cycle length (S2min), during S1S2 pacing. Longitudinal conduction velocity was increased in Gosr2+/− mice compared to WT at steady state- (76.44 vs. 67.00 cm/s) as well as at S2min (62.00 vs. 51.86 cm/s, p=0.039, n=10 and 9, resp.). Single cell patch-clamp studies revealed a shortened action potential duration at 90% repolarization at all pacing frequencies (390 vs 342 V/s at 2Hz, p=0.036) in isolated mid-LV cardiomyocytes of Gosr2+/− mice compared to WT. In addition, the maximal upstroke velocity was increased in Gosr2+/− mid-LV cardiomyocytes at frequencies of 6Hz and higher (390 vs 342 V/s at 6Hz, p=0.044). Conclusion Our findings identify Membrin as a novel interacting protein of NaV1.5 and a modulator of cardiac conduction. We propose that Membrin acts through ion channel trafficking or by modulating the posttranslational maturation of ion channels. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Leducq foundation

Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood

Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current (‘pump current’). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to −80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.

KV4.3 Expression Modulates NaV1.5 Sodium Current.

In cardiomyocytes, the voltage-gated transient outward potassium current (Ito) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the Ito carrying KV4.3 channel, have been associated with Brugada syndrome (BrS). While the role of Ito in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased Ito may directly affect cardiac conduction. However, the effects of an increased Ito on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of KV4.3 overexpression on NaV1.5 current and consequent sodium channel availability. We found that overexpression of KV4.3 protein in HEK293 cells stably expressing NaV1.5 (HEK293-NaV1.5 cells) significantly reduced NaV1.5 current density without affecting its kinetic properties. In addition, KV4.3 overexpression decreased AP upstroke velocity in HEK293-NaV1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of KV4.3 could not be explained by alterations in total NaV1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in KV4.3 current and concurrent decrease in NaV1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that KV4.3 directly impacts on NaV1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.

Resurgent sodium current promotes action potential firing in the avian auditory brainstem.

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

Eliminating spiral wave and spatiotemporal chaos in cardiac tissues by suppressing the rotation of spiral wave tip

The variation of the function of sodium channel in cardiomyocyte is associated with multiple cardiac diseases. Increasing sodium channel availability can effectively increase sodium influx, leading to enhanced cardiomyocyte excitability, prolonged action potential duration and late sodium current activity, which may cause ventricular arrhythmia. On the other hand, enhancing cardiomyocyte excitability can effectively increase the conduction velocity of the medium in the rotation center of spiral wave, which can restrain the rotation of spiral wave, leading to the disappearance of spiral wave. However, how to increase the excitability of cardiomyocytes while avoiding arrhythmias has not yet been explored so far. In this paper, we study how to regulate the changes of sodium current in cardiac myocytes to control spiral wave and spatiotemporal chaos in a two-dimensional cardiac tissues by using the Luo-Rudy phase I model. We propose such a sodium current control scheme:when the cell is excited, the regulation of sodium current begins. If the absolute value of sodium current obtained from the model equation is less than the absolute value of sodium current control threshold, the sodium current is simply equal to the control threshold of sodium current. In other cases, the absolute value of sodium current cannot exceed the maximum value without control. When the membrane potential rises over-5 mV, the sodium current evolves naturally. This method of regulating sodium current ensures that all cells have almost the same amplitude of sodium current, while without obviously changing the excitation-time. All cells thus have the same excitability under the control of sodium current, so that the excitation of cell is less affected by spiral wave tip. The numerical simulation results show that as long as the control threshold of sodium current reaches a critical value, the rotation of spiral wave tip is effectively suppressed, causing spiral wave to move out of the system boundary and spatiotemporal chaos to disappear after it has evolved into a spiral wave. If the absolute value of sodium current control threshold is large enough, the spiral wave and spatiotemporal chaos would also disappear through conductive block. These results can provide a new idea for antiarrhythmic therapy.

Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating.

Key points Sodium channels are critical for supporting fast action potentials in neurons; even mutations which cause small changes in sodium channel activity can have devastating consequences for the function of the nervous system.Alternative splicing also changes the activity of sodium channels, and while it is highly conserved, it is not known whether the functional role of this splicing is also conserved.Our data reveal that splicing has a highly conserved impact on the availability of sodium channels during trains of rapid stimulations, and suggest that in one mammalian channel, Nav1.1 encoded by SCN1A, the increased availability of one splice variant is detrimental.A model reproducing the effects of splicing on channel behaviour suggests that the voltage sensor in the first domain is a rate limiting step for release of the inactivation domain, and highlights the functional specialization of channel domains. Voltage‐gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) that splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals.

Calcium-activated SK channels control firing regularity by modulating sodium channel availability in midbrain dopamine neurons

Dopamine neurons in the substantia nigra pars compacta and ventral tegmental area regulate behaviours such as reward-related learning, and motor control. Dysfunction of these neurons is implicated in Schizophrenia, addiction to drugs, and Parkinson’s disease. While some dopamine neurons fire single spikes at regular intervals, others fire irregular single spikes interspersed with bursts. Pharmacological inhibition of calcium-activated potassium (SK) channels increases the variability in their firing pattern, sometimes also increasing the number of spikes fired in bursts, indicating that SK channels play an important role in maintaining dopamine neuron firing regularity and burst firing. However, the exact mechanisms underlying these effects are still unclear. Here, we develop a biophysical model of a dopamine neuron incorporating ion channel stochasticity that enabled the analysis of availability of ion channels in multiple states during spiking. We find that decreased firing regularity is primarily due to a significant decrease in the AHP that in turn resulted in a reduction in the fraction of available voltage-gated sodium channels due to insufficient recovery from inactivation. Our model further predicts that inhibition of SK channels results in a depolarisation of action potential threshold along with an increase in its variability.

Low Somatic Sodium Conductance Enhances Action Potential Precision in Time-Coding Auditory Neurons.

Proper hearing depends on analyzing temporal aspects of sounds with high precision. Auditory neurons that specialize in precise temporal information have a suite of unusual intrinsic properties, including nonovershooting action potentials and few sodium channels in the soma. However, it was not clear how low sodium channel availability in the soma influenced the temporal precision of action potentials initiated in the axon initial segment. We studied this using dynamic clamp to mimic sodium channels in the soma, which yielded normal, overshooting action potentials. Increasing somatic sodium conductance had major negative consequences: synaptic activity evoked action potentials with lower fidelity, and the precision of coincidence detection was degraded. Thus, low somatic sodium channel availability appears to enhance fidelity and temporal precision.

Refractoriness in human atria: Time and voltage dependence of sodium channel availability

BackgroundRefractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria. Methods and resultsSteady-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial fibrillation. The ex vivo measurements of action potential duration, effective refractory period and resting membrane potential were well-replicated in simulations using this new in silico model. Notably, the voltage threshold potential at which refractoriness was observed was not different between sinus rhythm and chronic atrial fibrillation tissues and was neither affected by changes in frequency (1 vs. 3Hz). ConclusionsOur results suggest a preferentially voltage-dependent, rather than time-dependent, effect with respect to refractoriness at physiologically relevant rates in human atria. However, as the resting membrane potential is hyperpolarized in chronic atrial fibrillation, the voltage-dependence of excitability dominates, profoundly increasing the risk for arrhythmia re-initiation and maintenance in fibrillating atria. Our results thereby highlight resting membrane potential as a potential target in pharmacological management of chronic atrial fibrillation.

Mechanism of Slow Inactivation in Prokaryotic Voltage-Gated Sodium Channel

Voltage-gated sodium channels (Nav) play a crucial role in electrically excitable cells by mediating the rapid influx of Na+ and thereby initiating the action potential in response to a depolarizing stimulus. In excitable cells, Nav activates and then undergoes fast and slow inactivation. Fast inactivation occurs within the time frame of a single action potential, whereas slow inactivation is produced by prolonged depolarization, mediating the availability of sodium channels over extended period of time. Mutations that alter slow inactivation are associated with several diseases such as hyperkalemic periodic analysis and long-QT syndrome. However, the molecular mechanism of slow inactivation and modulation by drug binding are still not completely known. In order to establish a structural framework of slow inactivation in molecular level, we used the prokaryotic homologue NavSP1 from Silicibacter pomeroyi as a model system. NavSP1 shares very similar overall architecture, activation, inactivation properties, and similar drug binding sites with the eukaryotic homologues and thereby proves to be an excellent model to be studied in native environment. We investigated how channel opening or the activation of voltage sensor initiates slow inactivation by evaluating the changes in conformational dynamics of the channel pore in the presence and absence of the voltage sensor. Our approach combined structural dynamics by EPR spectroscopy (CW and DEER methods) and functional studies by electrophysiological methods. Evaluation of the changes in the conformational dynamics and solvent accessibility in the turret and selectivity filter region show structural rearrangement of the outer pore region during slow inactivation.

Blockade of Calcium/Calmodulin-Dependent Kinase II (CaMKII) Induces Heterogeneous Depression of Conduction and Promotes Ischemic Ventricular Fibrillation (VF)

CaMKII regulates amongst many targets the sodium channel (INa), the primary determinant of cardiac excitability and conduction. Cellular studies diverge on the downstream effect of CaMKII on INa. Successful pharmacological therapy targeting CaMKII requires understanding how it regulates excitability and conduction in intact tissue, especially during myocardial ischemia when reduced sodium channel availability causes unsafe conduction. We recorded fluorescence of voltage-sensitive dye Di-4-ANEPPS in the left (LV) and right (RV) ventricles of Langendorff-perfused rabbit hearts (n=11) before and during 30 minutes of global ischemia, with and without perfusion of CaMKII blocker KN-93 (2.75 microM). We computed the local conduction delay (LCD, measured in ms/mm) and compared the 10-90% range of LCD values in the RV and LV at baseline and 4 minutes of ischemia. KN-93 caused heterogeneous slowing of conduction at baseline, which was further exacerbated by ischemia. Without major effect on the 10% level, KN-93 extended the 90% level of the LCD distribution (at baseline: 63% increase in LV; 211% increase in RV. At 4 minutes of ischemia: 230% increase in LV; 344% increase in RV; all p<0.05). During ischemia, KN-93 prompted the initiation (at 5.6±0.47 min of ischemia vs. 17.4±1.89 min in control, p<0.05) of VF through recurrent conduction block at a basal RV site of marked conduction depression. The “chemical phosphatase” butanedione monoxime (BDM, 20 mM) reduced almost threefold the KN-93 concentration required for depression of conduction and early ischemic VF. Conclusions: Constitutive activity of CaMKII is essential to maintain robust and uniform conduction in normal and acutely ischemic heart. CaMKII block is pro-arrhythmic during ischemia. The increased sensitivity to KN-93 in the presence of BDM suggests a delicate balance between kinase and phosphatase activity in regulation of cardiac conduction.

Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

SummaryFunctional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells.

Antiarrhythmic Mechanisms of SK Channel Inhibition in the Rat Atrium.

SK channels have functional importance in the cardiac atrium of many species, including humans. Pharmacological blockage of SK channels has been reported to be antiarrhythmic in animal models of atrial fibrillation; however, the exact antiarrhythmic mechanism of SK channel inhibition remains unclear. We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability, thereby prolonging the effective refractory period and slowing the conduction velocity (CV). We therefore aimed at elucidating these properties of SK channel inhibition and the underlying antiarrhythmic mechanisms using microelectrode action potential (AP) recordings and CV measurements in isolated rat atrium. Automated patch clamping and two-electrode voltage clamp were used to access INa and IK,ACh, respectively. The SK channel inhibitor N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) exhibited antiarrhythmic effects. ICA prevented electrically induced runs of atrial fibrillation in the isolated right atrium and induced atrial postrepolarization refractoriness and depolarized RMP. Moreover, ICA (1-10 μM) was found to slow CV; however, because of a marked prolongation of effective refractory period, the calculated wavelength was increased. Furthermore, at increased pacing frequencies, SK channel inhibition by ICA (10-30 μM) demonstrated prominent depression of other sodium channel-dependent parameters. ICA did not inhibit IK,ACh, but at concentrations above 10 μM, ICA use dependently inhibited INa. SK channel inhibition modulates multiple parameters of AP. It prolongs the AP duration and shifts the RMP towards more depolarized potentials through direct ISK block. This indirectly leads to sodium channel inhibition through accumulation of state dependently inactivated channels, which ultimately slows conduction and decreases excitability. However, a contribution from a direct sodium channel inhibition cannot be ruled. We here propose that the primary antiarrhythmic mechanism of SK channel inhibition is through direct potassium channel block and through indirect sodium channel inhibition.

Delayed afterdepolarizations generate both triggers and a vulnerable substrate promoting reentry in cardiac tissue

Delayed afterdepolarizations (DADs) have been well characterized as arrhythmia triggers, but their role in generating a tissue substrate vulnerable to reentry is not well understood. The purpose of this study was to test the hypothesis that random DADs can self-organize to generate both an arrhythmia trigger and a vulnerable substrate simultaneously in cardiac tissue as a result of gap junction coupling. Computer simulations in 1-dimensional cable and 2-dimensional tissue models were performed. The cellular DAD amplitude was varied by changing the strength of sarcoplasmic reticulum calcium release. Random DAD latency and amplitude in different cells were simulated using gaussian distributions. Depending on the strength of spontaneous sarcoplasmic reticulum calcium release and other conditions, random DADs in cardiac tissue resulted in the following behaviors: (1) triggered activity (TA); (2) a vulnerable tissue substrate causing unidirectional conduction block and reentry by inactivating sodium channels; (3) both triggers and a vulnerable substrate simultaneously by generating TA in regions next to regions with subthreshold DADs susceptible to unidirectional conduction block and reentry. The probability of the latter 2 behaviors was enhanced by reduced sodium channel availability, reduced gap junction coupling, increased tissue heterogeneity, and less synchronous DAD latency. DADs can self-organize in tissue to generate arrhythmia triggers, a vulnerable tissue substrate, and both simultaneously. Reduced sodium channel availability and gap junction coupling potentiate this mechanism of arrhythmias, which are relevant to a variety of heart disease conditions.

Sensitization of enteric neurons to morphine by HIV-1 Tat protein.

Gastrointestinal (GI) dysfunction is a major cause of morbidity in acquired immunodeficiency syndrome (AIDS). HIV-1-induced neuropathogenesis is significantly enhanced by opiate abuse, which increases proinflammatory chemokine/cytokine release, the production of reactive species, glial reactivity, and neuronal injury in the central nervous system. Despite marked interactions in the gut, little is known about the effects of HIV-1 in combination with opiate use on the enteric nervous system. To explore HIV-opiate interactions in myenteric neurons, the effects of Tat ± morphine (0.03, 0.3, and 3 μM) were examined in isolated neurons from doxycycline- (DOX-) inducible HIV-1 Tat(1-86) transgenic mice or following in vitro Tat 100 nM exposure (>6 h). Current clamp recordings demonstrated increased neuronal excitability in neurons of inducible Tat(+) mice (Tat+/DOX) compared to control Tat-/DOX mice. In neurons from Tat+/DOX, but not from Tat-/DOX mice, 0.03 μM morphine significantly reduced neuronal excitability, fast transient and late long-lasting sodium currents. There was a significant leftward shift in V(0.5) of inactivation following exposure to 0.03 μM morphine, with a 50% decrease in availability of sodium channels at -100 mV. Similar effects were noted with in vitro Tat exposure in the presence of 0.3 μM morphine. Additionally, GI motility was significantly more sensitive to morphine in Tat(+) mice than Tat(-) mice. Overall, these data suggest that the sensitivity of enteric neurons to morphine is enhanced in the presence of Tat. Opiates and HIV-1 may uniquely interact to exacerbate the deleterious effects of HIV-1-infection and opiate exposure on GI function.

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst-firing patterns that give rise to elevated Ca(2+)-influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels. Action potential (AP) firing in mouse chromaffin cells (MCCs) is mainly sustained by Cav1.3 L-type channels that drive BK and SK currents and regulate the pacemaking cycle. As secretory units, CCs optimally recruit Ca(2+) channels when stimulated, a process potentially dependent on the modulation of the AP waveform. Our previous work has shown that a critical determinant of AP shape is voltage-gated sodium channel (Nav) channel availability. Here, we studied the contribution of Nav channels to firing patterns and AP shapes at rest (-50 mV) and upon stimulation (-40 mV). Using quantitative RT-PCR and immunoblotting, we show that MCCs mainly express tetrodotoxin (TTX)-sensitive, fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no Na(+) current during slow ramp depolarizations. Time constants and the percentage of recovery from fast inactivation and slow entry into closed-state inactivation are similar to that of brain Nav1.3 and Nav1.7 channels. The fraction of available Nav channels is reduced by half after 10 mV depolarization from -50 to -40 mV. This leads to low amplitude spikes and a reduction in repolarizing K(+) currents inverting the net current from outward to inward during the after-hyperpolarization. When Nav channel availability is reduced by up to 20% of total, either by TTX block or steady depolarization, a switch from tonic to burst firing is observed. The spontaneous occurrence of high frequency bursts is rare under control conditions (14% of cells) but leads to major Ca(2+)-entry and increased catecholamine release. Thus, Nav1.3/Nav1.7 channel availability sets the AP shape, burst-firing initiation and regulates catecholamine secretion in MCCs. Nav channel inactivation becomes important during periods of high activity, mimicking stress responses.

Abstract 14019: Electrophysiological Effects of the Late Sodium Current Inhibitor GS967 in Scn5a -1798insD Mouse and Human SCN5A -1795insD iPSC-derived Cardiomyocytes

Background and Aim: Selective inhibition of cardiac late sodium current (I Na,L ) is an emerging target in the treatment of ventricular arrhythmias. The electrophysiological effects of GS967, a potent I Na,L inhibitor, were investigated in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from human SCN5A -1795insD induced pluripotent stem cells (iPSC-CMs) and mice carrying the homologous mutation Scn5a -1798insD. Methods and Results: On patch-clamp analysis, isolated mouse Scn5a -1798insD cardiomyocytes and human SCN5A -1795insD iPSC-CMs showed decreased peak I Na and action potential (AP) upstroke velocity (Vmax) and increased I Na,L and AP duration at 90% repolarization (APD 90 ) as compared to wild-type. GS967 (50-300 nM) significantly decreased APD 90 in mouse Scn5a -1798insD cardiomyocytes by 8±2% (mean±SEM) at 50 nM (n=7), 13±3% at 100 nM (n=11) and 20±5% at 300 nM (n=6) (all p &lt;0.01 vs. control), without affecting Vmax. GS967 (300 nM) selectively inhibited I NaL in mouse Scn5a -1798insD cardiomyocytes (GS967-sensitive current of 0.7±0.1 pA/pF, n=6), but had no effect on peak I Na . Furthermore, GS967 (100 nM) suppressed fast (5 Hz) pacing-induced afterpotentials and triggered activity. In human SCN5A -1795insD iPSC-CMs (n=6), GS967 (300 nM) significantly reduced APD 90 without affecting the resting membrane potential or Vmax. In Langendorff-perfused, isolated mouse Scn5a -1798insD hearts (n=5), GS967 (300 nM) had no effect on ventricular activation time or conduction velocity (as assessed by epicardial mapping). Conclusion: Selective inhibition of I NaL by GS967 attenuated AP prolongation and prevented pro-arrhythmic activity in mouse Scn5a -1798insD cardiomyocytes and human SCN5A-1795insD iPSC-CMs, thus suppressing the gain-of-function features of this overlap syndrome mutation. Importantly, these beneficial actions of GS967 occurred in the absence of deleterious effects on sodium channel availability or cardiac conduction, despite a pre-existing decrease in peak I Na . Thus, selective inhibition of I Na,L constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced I NaL , even in overlap syndromes whereby peak I Na is decreased.

The role of slow and persistent TTX-resistant sodium currents in acute tumor necrosis factor-α-mediated increase in nociceptors excitability.

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes.