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Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.
Copyright © 2015 Elsevier Inc. All rights reserved.
Two-pore domain K+ (K2P) channels play an important role in tuning β-cell glucose-stimulated insulin secretion (GSIS). The K2P channel TWIK-related alkaline pH-activated K2P (TALK)-1 is linked to type 2 diabetes risk through a coding sequence polymorphism (rs1535500); however, its physiological function has remained elusive. Here, we show that TALK-1 channels are expressed in mouse and human β-cells, where they serve as key regulators of electrical excitability and GSIS. We find that the rs1535500 polymorphism, which results in an alanine-to-glutamate substitution in the C-terminus of human TALK-1, increases channel activity. Genetic ablation of TALK-1 results in β-cell membrane potential depolarization, increased islet Ca2+ influx, and enhanced second-phase GSIS. Moreover, mice lacking TALK-1 channels are resistant to high-fat diet-induced elevations in fasting glycemia. These findings reveal TALK-1 channels as important modulators of second-phase insulin secretion and suggest a clinically relevant mechanism for rs1535500, which may increase type 2 diabetes risk by limiting GSIS.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.
KEY POINTS - Atrial fibrillation is often initiated and perpetuated by abnormal electrical pulses repetitively originating from regions outside the heart's natural pacemaker. In this study we examined the causal role of abnormal calcium releases from the sarcoplasmic reticulum in producing repetitive electrical discharges in atrial cells and tissues. Calsequestrin2 is a protein that stabilizes the closed state of calcium release channels, i.e. the ryanodine receptors. In the atria from mice predisposed to abnormal calcium releases secondary to the absence of calsequestrin2, we observed abnormal repetitive electrical discharges that may lead to atrial fibrillation. Here, we report a novel pathological rhythm generator. Specifically, abnormal calcium release leads to electrical activation, which in turn results in another abnormal calcium release. This process repeats itself and thus sustains the repetitive electrical discharges. These results suggest that improving the stability of ryanodine receptors might be useful to treat atrial fibrillation.
ABSTRACT - Aberrant diastolic calcium (Ca) release due to leaky ryanodine receptors (RyR2s) has been recently associated with atrial fibrillation (AF) and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, it remains unclear how diastolic Ca release contributes to the rising of rapid repetitive focal activity, which is considered as a common AF triggering mechanism. To address this question, we conducted simultaneous voltage/Ca optical mapping in atrial tissue and one-/two-dimensional confocal imaging in atrial tissue and myocytes from wild-type (WT, n = 15) and CPVT mice lacking calsequestrin 2 (Casq2(-/-), n = 45), which promotes diastolic Ca release. During β-adrenergic stimulation (100 nM isoproterenol), only Casq2(-/-) atrial myocytes showed pacing-induced self-sustained repetitive activity (31 ± 21 s vs. none in WT). Importantly, in atrial tissue, this repetitive activity could translate to Ca-dependent focal arrhythmia. Ectopic action potential (AP) firing during repetitive activity occurred only when diastolic Ca release achieved a sufficient level of synchronization. The AP, in turn, synchronized subsequent diastolic Ca release by temporally aligning multiple sources of Ca waves both within individual myocytes and throughout the atrial tissue. This alternating interplay between AP and diastolic Ca release perpetuates the self-sustaining repetitive activity. In fact, pharmacological disruption of synchronized diastolic Ca release (by ryanodine) prevented aberrant APs; and vice versa, the inhibition of AP (by TTX or 0 Na, 0 Ca solution) de-synchronized diastolic Ca release. Taken together, these results suggest that a cyclical interaction between synchronized diastolic Ca release and AP forms a pathological rhythm generator that is involved in Ca-dependent atrial arrhythmias in CPVT.
© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.
AIMS - To test the hypothesis that vulnerability to atrial fibrillation (AF) is associated with rare coding sequence variation in the SCN10A gene, which encodes the voltage-gated sodium channel isoform NaV1.8 found primarily in peripheral nerves and to identify potentially disease-related mechanisms in high-priority rare variants using in-vitro electrophysiology.
METHODS AND RESULTS - We re-sequenced SCN10A in 274 patients with early onset AF from the Vanderbilt AF Registry to identify rare coding variants. Engineered variants were transiently expressed in ND7/23 cells and whole-cell voltage clamp experiments were conducted to elucidate their functional properties. Resequencing SCN10A identified 18 heterozygous rare coding variants (minor allele frequency ≤1%) in 18 (6.6%) AF probands. Four probands were carriers of two rare variants each and 14 were carriers of one coding variant. Based on evidence of co-segregation, initial assessment of functional importance, and presence in ≥1 AF proband, three variants (417delK, A1886V, and the compound variant Y158D-R814H) were selected for functional studies. The 417delK variant displayed near absent current while A1886V and Y158D-R814H exhibited enhanced peak and late (INa-L) sodium currents; both Y158D and R818H individually contributed to this phenotype.
CONCLUSION - Rare SCN10A variants encoding Nav1.8 were identified in 6.6% of patients with early onset AF. In-vitro electrophysiological studies demonstrated profoundly altered function in 3/3 high-priority variants. Collectively, these data strongly support the hypothesis that rare SCN10A variants may contribute to AF susceptibility.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: firstname.lastname@example.org.
Dopaminergic innervation of the extended amygdala regulates anxiety-like behavior and stress responsivity. A portion of this dopamine input arises from dopamine neurons located in the ventral lateral periaqueductal gray (vlPAG) and rostral (RLi) and caudal linear nuclei of the raphe (CLi). These neurons receive substantial norepinephrine input, which may prime them for involvement in stress responses. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase promoter, we explored the physiology and responsiveness to norepinephrine of these neurons. We find that RLi dopamine neurons differ from VTA dopamine neurons with respect to membrane resistance, capacitance and the hyperpolarization-activated current, Ih. Further, we found that norepinephrine increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) on RLi dopamine neurons. This effect was mediated through the α1 adrenergic receptor (AR), as the actions of norepinephrine were mimicked by the α1-AR agonist methoxamine and blocked by the α1-AR antagonist prazosin. This action of norepinephrine on sEPSCs was transient, as it did not persist in the presence of prazosin. Methoxamine also increased the frequency of miniature EPSCs, indicating that the α1-AR action on glutamatergic transmission likely has a presynaptic mechanism. There was also a modest decrease in sEPSC frequency with the application of the α2-AR agonist UK-14,304. These studies illustrate a potential mechanism through which norepinephrine could recruit the activity of this population of dopaminergic neurons.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Calcium entry through voltage-dependent Ca(2+) channels (VDCCs) is required for pancreatic β-cell insulin secretion. The 2-pore-domain acid-sensitive potassium channel (TASK-1) regulates neuronal excitability and VDCC activation by hyperpolarizing the plasma membrane potential (Δψp); however, a role for pancreatic β-cell TASK-1 channels is unknown. Here we examined the influence of TASK-1 channel activity on the β-cell Δψp and insulin secretion during secretagogue stimulation. TASK-1 channels were found to be highly expressed in human and rodent islets and localized to the plasma membrane of β-cells. TASK-1-like currents of mouse and human β-cells were blocked by the potent TASK-1 channel inhibitor, A1899 (250nM). Although inhibition of TASK-1 currents did not influence the β-cell Δψp in the presence of low (2mM) glucose, A1899 significantly enhanced glucose-stimulated (14mM) Δψp depolarization of human and mouse β-cells. TASK-1 inhibition also resulted in greater secretagogue-stimulated Ca(2+) influx in both human and mouse islets. Moreover, conditional ablation of mouse β-cell TASK-1 channels reduced K2P currents, increased glucose-stimulated Δψp depolarization, and augmented secretagogue-stimulated Ca(2+) influx. The Δψp depolarization caused by TASK-1 inhibition resulted in a transient increase in glucose-stimulated mouse β-cell action potential (AP) firing frequency. However, secretagogue-stimulated β-cell AP duration eventually increased in the presence of A1899 as well as in β-cells without TASK-1, causing a decrease in AP firing frequency. Ablation or inhibition of mouse β-cell TASK-1 channels also significantly enhanced glucose-stimulated insulin secretion, which improved glucose tolerance. Conversely, TASK-1 ablation did not perturb β-cell Δψp, Ca(2+) influx, or insulin secretion under low-glucose conditions (2mM). These results reveal a glucose-dependent role for β-cell TASK-1 channels of limiting glucose-stimulated Δψp depolarization and insulin secretion, which modulates glucose homeostasis.
We compared the effects of three missense mutations in the GABAA receptor γ2 subunit on GABAA receptor assembly, trafficking and function in HEK293T cells cotransfected with α1, β2, and wildtype or mutant γ2 subunits. The mutations R82Q and P83S were identified in families with genetic epilepsy with febrile seizures plus (GEFS+), and N79S was found in a single patient with generalized tonic-clonic seizures (GTCS). Although all three mutations were located in an N-terminal loop that contributes to the γ+/β- subunit-subunit interface, we found that each mutation impaired GABAA receptor assembly to a different extent. The γ2(R82Q) and γ2(P83S) subunits had reduced α1β2γ2 receptor surface expression due to impaired assembly into pentamers, endoplasmic reticulum (ER) retention and degradation. In contrast, γ2(N79S) subunits were efficiently assembled into GABAA receptors with only minimally altered receptor trafficking, suggesting that N79S was a rare or susceptibility variant rather than an epilepsy mutation. Increased structural variability at assembly motifs was predicted by R82Q and P83S, but not N79S, substitution, suggesting that R82Q and P83S substitutions were less tolerated. Membrane proteins with missense mutations that impair folding and assembly often can be "rescued" by decreased temperatures. We coexpressed wildtype or mutant γ2 subunits with α1 and β2 subunits and found increased surface and total levels of both wildtype and mutant γ2 subunits after decreasing the incubation temperature to 30°C for 24h, suggesting that lower temperatures increased GABAA receptor stability. Thus epilepsy-associated mutations N79S, R82Q and P83S disrupted GABAA receptor assembly to different extents, an effect that could be potentially rescued by facilitating protein folding and assembly.
Copyright © 2014 Elsevier Inc. All rights reserved.
The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca(2+)) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca(2+) dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca(2+) store depletions and membrane depolarization with ER store-operated Ca(2+) entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca(2+) transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca(2+) release and MT uptake on bulk cytosolic Ca(2+) levels because persistent elevations of free intracellular Ca(2+) are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca(2+) and the resulting frequencies of ER Ca(2+) store depletions, as well as the sarco-endoplasmic reticulum Ca(2+) ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca(2+) depletion frequencies than protection of the cytosol from elevated free Ca(2+), whereas the SERCA pump is more relevant to containing cytosolic Ca(2+) elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca(2+) release, subsequent MT uptake, and eventual activation of ER store-operated Ca(2+) entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types.
Copyright © 2014 the American Physiological Society.
Of the five muscarinic receptor subtypes, the M5 receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5 in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5 receptor. We then used VU0238429, along with M5 knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+ mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5 knock-out mice. Activation of M5 also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5 increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5 with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5 can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5 receptors on SNc neurons leads to increased neuronal firing, activation of M5 receptors in the striatum induces an inhibition in dopamine release.