Regulation of the voltage-dependent sodium channel NaV1.1 by AKT1.

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.

Modulation of acid-sensing ion channels by hydrogen sulfide.

Acid-sensing ion channels (ASICs) have been implicated in many physiological and patho-physiological processes like synaptic plasticity, inflammation, pain perception, stroke-induced brain damage and, drug-seeking behaviour. Although ASICs have been shown to be modulated by gasotransmitters like nitric oxide (NO), their regulation by hydrogen sulfide (H2S) is not known. Here, we present strong evidence that H2S potentiates ASICs-mediated currents. Low pH-induced current in Chinese hamster ovary (CHO) cells, expressing homomeric either ASIC1a, ASIC2a or ASIC3, increased significantly by an H2S donor NaHS. The effect was reversed by washing the cells with NaHS-free external solution of pH 7.4. MTSES, a membrane impermeable cysteine thiol-modifier failed to abrogate the effect of NaHS on ASIC1a, suggesting that the target cysteine residues are not in the extracellular region of the channel. The effect of NaHS is not mediated through NO, as the basal NO level in cells did not change following NaHS application. This previously unknown mechanism of ASICs-modulation by H2S adds a new dimension to the ASICs in health and disease.

A selective NaV1.1 activator with potential for treatment of Dravet syndrome epilepsy.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (NaV1.1), lead to DS. NaV1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principle mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional NaV1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate NaV1.1, and showed that selective activation of NaV1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system, and examined its selectivity against a panel of human NaV subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of NaV1.1 and NaV1.3 (EC50 ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel's voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

Multiple regulatory actions of 2-guanidine-4-methylquinazoline (GMQ), an agonist of acid-sensing ion channel type 3, on ionic currents in pituitary GH3 cells and in olfactory sensory (Rolf B1.T) neurons.

GMQ (2-guanidine-4-methylquinazoline or N-(4-methyl-2-quinazolinyl)-guanidine hydrochloride), an agonist of acid-sensing ion channel type 3, has been increasingly used for in vivo studies of alternations in nociceptic behavior. In this study, we tried to investigate whether GMQ has any possible effect on other types of ion channels. Addition of GMQ to pituitary GH3 cells raised the amplitude of Ca2+-activated K+ currents (IK(Ca)), which was reversed by verruculogen or PF1022A, but not by TRAM-39. Under inside-out current recordings, addition of GMQ into bath enhanced the probability of large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 0.95 µM. The activation curve of BKCa channels during exposure to GMQ shifted to a lower depolarized potential, with no change in the gating charge of the curve; however, there was a reduction of free energy for channel activation in its presence. As cells were exposed to GMQ, the amplitude of ion currents were suppressed, including delayed rectifying K+ current, voltage-gated Na+ and L-type Ca2+ currents. In Rolf B1.T olfactory sensory neuron, addition of GMQ was able to induce inward current and to suppress peak INa. Taken together, findings from these results indicated that in addition to the activation of ASIC3 channels, this compound might directly produce additional actions on various types of ion channels. Caution should be taken in the interpretation of in vivo experimental results when GMQ or other structurally similar compounds are used as targets to characterize the potential functions of ASIC3 channels.

Veratridine-sensitive Na+ channels regulate human sperm fertilization capacity.

AIMS: The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca2+, Na+, K+ and H+ ions in the sperm cytoplasm. Ion channels are key regulators of the sperm membrane potential, cytoplasmic Ca2+ and intracellular pH (pHi), which leads to regulate motility, capacitation, acrosome reaction and other physiological processes crucial for successful fertilization. Expression of epithelial sodium channels (ENaC) and voltage-gated sodium channels (Nav) in human spermatozoa has been reported, but the role of Na+ fluxes sodium channels in the regulation of sperm cell function remains poorly understood. In this context, we aimed to analyze the physiological role of Nav channels in human sperm. MAIN METHODS: Motility and hyperactivation analysis was conducted by CASA analysis. Flow cytometry and spectrophotometry approaches were carried out to measure Capacitation, Acrosome reaction, immunohistochemistry for Tyr-residues phosporylation, [Ca2+]i levels and membrane potential. KEY FINDINGS: Functional studies showed that veratridine, a voltage-gated sodium channel activator, increased sperm progressive motility without producing hyperactivation while the Nav antagonist lidocaine did induce hyperactivated motility. Veratridine increased protein tyrosine phosphorylation, an event occurring during capacitation, and its effects were inhibited in the presence of lidocaine and tetrodotoxin. Veratridine had no effect on the acrosome reaction by itself, but was able to block the progesterone-induced acrosome reaction. Moreover, veratridine caused a membrane depolarization and modified the effect of progesterone on [Ca2+]i and sperm membrane potential. SIGNIFICANCE: Our results suggest that veratridine-sensitive Nav channels are involved on human sperm fertility acquisition regulating motility, capacitation and the progesterone-induced acrosome reaction in human sperm.

Modeling of action potential generation in NG108-15 cells.

In order to explore the possibility of identifying toxins based on their effect on the shape of action potentials, we created a computer model of the action potential generation in NG108-15 cells (a neuroblastoma/glioma hybrid cell line). To generate the experimental data for model validation, voltage-dependent sodium, potassium and high-threshold calcium currents, as well as action potentials, were recorded from NG108-15 cells with conventional whole-cell patch-clamp methods. Based on the classic Hodgkin-Huxley formalism and the linear thermodynamic description of the rate constants, ion-channel parameters were estimated using an automatic fitting method. Utilizing the established parameters, action potentials were generated using the Hodgkin-Huxley formalism and were fitted to the recorded action potentials. To demonstrate the applicability of the method for toxin detection and discrimination, the effect of tetrodotoxin (a sodium channel blocker) and tefluthrin (a pyrethroid that is a sodium channel opener) were studied. The two toxins affected the shape of the action potentials differently, and their respective effects were identified based on the predicted changes in the fitted parameters.

Antiarrhythmic effects of (-)-epicatechin-3-gallate, a novel sodium channel agonist in cultured neonatal rat ventricular myocytes.

(-)-Epicatechin-3-gallate (ECG), a polyphenol extracted from green tea, has been proposed as an effective compound for improving cardiac contractility. However, the therapeutic potential of ECG on the treatment of arrhythmia remains unknown. We investigated the direct actions of ECG on the modulation of ion currents and cardiac cell excitability in the primary culture of neonatal rat ventricular myocyte (NRVM), which is considered a hypertrophic model for analysis of myocardial arrhythmias. By using the whole-cell patch-clamp configurations, we found ECG enhanced the slowly inactivating component of voltage-gated Na(+) currents (I(Na)) in a concentration-dependent manner (0.1-100 µM) with an EC(50) value of 3.8 µM. ECG not only shifted the current-voltage relationship of peak I(Na) to the hyperpolarizing direction but also accelerated I(Na) recovery kinetics. Working at a concentration level of I(Na) enhancement, ECG has no notable effect on voltage-gated K(+) currents and L-type Ca(2+) currents. With culture time increment, the firing rate of spontaneous action potential (sAP) in NRVMs was gradually decreased until spontaneous early after-depolarization (EAD) was observed after about one week culture. ECG increased the firing rate of normal sAP about two-fold without waveform alteration. Interestingly, the bradycardia-dependent EAD could be significantly restored by ECG in fast firing rate to normal sAP waveform. The expression of dominant cardiac sodium channel subunit, Nav1.5, was consistently detected throughout the culture periods. Our results reveal how ECG, the novel I(Na) agonist, may act as a promising candidate in clinical applications on cardiac arrhythmias.