GPER mediated estrogenic amelioration of sodium channel dysfunction in stressed human induced pluripotent stem cell-derived cardiomyocytes

Stress-induced excessive activation of the adrenergic system or changes in estrogen levels promote the occurrence of arrhythmias. Sodium channel, a responder to β-adrenergic stimulation, is involved in stress-induced cardiac electrophysiological abnormalities. However, it has not been established whether estrogen regulates sodium channels during acute stress. Our study aimed to explore whether voltage-gated sodium channels play roles in the rapid regulation of various concentrations of estrogen in stressed human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and reveal the possible mechanism of estrogen signaling pathway modulating stress. An isoproterenol-induced stress model of hiPSC-CMs was pre-incubated with β-Estradiol at different concentrations (0.01 nmol/L, 1 nmol/L, and 100 nmol/L). Action potential (AP) and sodium currents were detected by patch clamp. The G protein-coupled estrogen receptor (GPER)-specific effect was determined with agonists G1, antagonists G15 and small interfering RNA. β-Estradiol at concentrations of 0.01 nmol/L, 1 nmol/L, and 100 nmol/L increased the peak sodium current and prolonged AP duration (APD) at 1 nmol/L. Stress increased peak sodium current, late sodium current, and shortened APD. The effects of stress on sodium currents and APD were eliminated by β-Estradiol. Activation of GPER by G1 exhibited similar effects as β-Estradiol, while inhibition of GPER with G15 and small interfering RNA ameliorated estrogenic actions. Estrogen, antagonized the stress-related abnormal electrical activity, and through GPER alleviated sodium channel dysfunctions in stress state in hiPSC-CMs. These results provide a novel mechanism through which estrogenic rapid signaling against stress by regulating ion channels.

Persistent sodium currents in SCN1A developmental and degenerative epileptic dyskinetic encephalopathy

Pathogenic variants in the voltage-gated sodium channel gene (SCN1A) are amongst the most common genetic causes of childhood epilepsies. There is considerable heterogeneity in both the types of causative variants and associated phenotypes; a recent expansion of the phenotypic spectrum of SCN1A associated epilepsies now includes an early onset severe developmental and epileptic encephalopathy with regression and a hyperkinetic movement disorder. Herein, we report a female with a developmental and degenerative epileptic-dyskinetic encephalopathy, distinct and more severe than classic Dravet syndrome. Clinical diagnostics indicated a paternally inherited c.5053G>T; p. A1685S variant of uncertain significance in SCN1A. Whole-exome sequencing detected a second de novo mosaic (18%) c.2345G>A; p. T782I likely pathogenic variant in SCN1A (maternal allele). Biophysical characterisation of both mutant channels in a heterologous expression system identified gain-of-function effects in both, with a milder shift in fast inactivation of the p. A1685S channels; and a more severe persistent sodium current in the p. T782I. Using computational models, we show that large persistent sodium currents induce hyperexcitability in individual cortical neurons, thus relating the severe phenotype to the empirically quantified sodium channel dysfunction. These findings further broaden the phenotypic spectrum of SCN1A associated epilepsies and highlight the importance of testing for mosaicism in epileptic encephalopathies. Detailed biophysical evaluation and computational modelling further highlight the role of gain-of-function variants in the pathophysiology of the most severe phenotypes associated with SCN1A.

Dopaminergic Inhibition of Na+ Currents in Vestibular Inner Ear Afferents

Inner ear hair cells form synapses with afferent terminals and afferent neurons carry signals as action potentials to the central nervous system. Efferent neurons have their origins in the brainstem and some make synaptic contact with afferent dendrites beneath hair cells. Several neurotransmitters have been identified that may be released from efferent terminals to modulate afferent activity. Dopamine is a candidate efferent neurotransmitter in both the vestibular and auditory systems. Within the cochlea, activation of dopamine receptors may reduce excitotoxicity at the inner hair cell synapse via a direct effect of dopamine on afferent terminals. Here we investigated the effect of dopamine on sodium currents in acutely dissociated vestibular afferent calyces to determine if dopaminergic signaling could also modulate vestibular responses. Calyx terminals were isolated along with their accompanying type I hair cells from the cristae of gerbils (P15-33) and whole cell patch clamp recordings performed. Large transient sodium currents were present in all isolated calyces; compared to data from crista slices, resurgent Na+ currents were rare. Perfusion of dopamine (100 μM) in the extracellular solution significantly reduced peak transient Na+ currents by approximately 20% of control. A decrease in Na+ current amplitude was also seen with extracellular application of the D2 dopamine receptor agonist quinpirole, whereas the D2 receptor antagonist eticlopride largely abolished the response to dopamine. Inclusion of the phosphatase inhibitor okadaic acid in the patch electrode solution occluded the response to dopamine. The reduction in calyx sodium current in response to dopamine suggests efferent signaling through D2 dopaminergic receptors may occur via common mechanisms to decrease excitability in inner ear afferents.

Differential roles of trithorax protein MLL-1 in regulating neuronal Ion channels

Repressive regulation of potassium channel genes by Polycomb group (PcG) proteins contributes to PcG protein-mediated neuroprotection against neuronal ischemic injury, as seen in an ischemic stroke. Here we asked the question whether Trithorax group (TrxG) proteins, the antagonistic partners of PcG proteins (i.e, epigenetic activators targeting the same genes) may also regulate potassium channels. Results of patch-clamp studies on cultured neuronal cells showed that inhibition of TrxG protein MLL-1 led to an increase in potassium channel activity, an unexpected effect for a presumed gene activator. In contrast, decreased sodium currents were observed with MLL-1 inhibition. Increased or decreased levels of potassium channel protein Kv2.1 or sodium channel protein Nav1.2, respectively, were seen with MLL-1 inhibition, as determined by immunocytochemistry. These results, for the first time, demonstrate an involvement of TrxG protein MLL-1 in regulating neuronal ion channels, potentially repressing potassium channel genes.

Abstract P272: Mechanisms By Which Aldosterone And Insulin Stimulate Endothelial Cell Sodium Currents.

Aldosterone and insulin are known to increase the activity of the epithelial Na + channel (ENaC) in tubular epithelial cells through multiple mechanisms involving both transcriptional and post-transcriptional events. Such studies have implicated a key role for serum and glucocorticoid-stimulated kinase 1 (SGK-1) in determining ENaC activity. Comparatively less information is available regarding such regulation of the endothelial cell (EC) Na+ channel (EnNaC). The importance of such knowledge relates to prior studies having shown that EnNaC contributes to cardiovascular stiffening in states of hyperaldosteronism and hyperinsulinemia/insulin resistance. We hypothesized that aldosterone and insulin converge on SGK-1 to increase EnNaC activity. To determine how EnNaC is affected by insulin and aldosterone pulmonary ECs were isolated from wild-type mice and global SGK-1 deficient mice and held under conditions of short-term tissue culture. ECs were subsequently exposed to aldosterone (10 nM), insulin (100 nM) or DMSO control for 24 hr and whole cell EnNaC currents measured by patch clamp. Both chronic aldosterone and insulin increased Na + currents in ECs from wild-type mice, while in cells from SGK-1-/- mice the response to aldosterone was significantly blunted. However, insulin continued to increase EnNaC activity in SGK-1-/- mice. Acute insulin treatment (10 mins) showed a concentration-dependent (10 – 100 nM) increase in EC Na + currents in both SGK+/+ and SGK-/- mice which was prevented by PI3 kinase inhibition with LY294002 (30 uM). Thus, in ECs aldosterone-mediated activation of EnNaC is dependent on SGK-1 while insulin exerts a stimulatory effect via PI3 kinase, even in the absence of SGK-1.

Anti–contactin-1 Antibodies Affect Surface Expression and Sodium Currents in Dorsal Root Ganglia

Background and ObjectivesAs autoantibodies to contactin-1 from patients with chronic inflammatory demyelinating polyradiculoneuropathy not only bind to the paranodes where they are supposed to cause conduction failure but also bind to other neuronal cell types, we aimed to investigate the effect of anti–contactin-1 autoantibodies on contactin-1 surface expression in cerebellar granule neurons, dorsal root ganglion neurons, and contactin-1–transfected human embryonic kidney 293 cells.MethodsImmunocytochemistry including structured illumination microscopy and immunoblotting was used to determine expression levels of contactin-1 and/or sodium channels after long-term exposure to autoantibodies from 3 seropositive patients. For functional analysis of sodium channels, whole-cell recordings of sodium currents were performed on dorsal root ganglion neurons incubated with anti–contactin-1 autoantibodies.ResultsWe found a reduction in contactin-1 expression levels on dorsal root ganglion neurons, cerebellar granule neurons, and contactin-1–transfected human embryonic kidney 293 cells and decreased dorsal root ganglion sodium currents after long-term exposure to anti–contactin-1 autoantibodies. Sodium channel density did not decrease.DiscussionOur results demonstrate a direct effect of anti–contactin-1 autoantibodies on the surface expression of contactin-1 and sodium currents in dorsal root ganglion neurons. This may be the pathophysiologic correlate of sensory ataxia reported in these patients.

Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.