EpiPro, a Novel, Synthetic, Activity-Regulated Promoter That Targets Hyperactive Neurons in Epilepsy for Gene Therapy Applications.

Epileptogenesis is characterized by intrinsic changes in neuronal firing, resulting in hyperactive neurons and the subsequent generation of seizure activity. These alterations are accompanied by changes in gene transcription networks, first with the activation of early-immediate genes and later with the long-term activation of genes involved in memory. Our objective was to engineer a promoter containing binding sites for activity-dependent transcription factors upregulated in chronic epilepsy (EpiPro) and validate it in multiple rodent models of epilepsy. First, we assessed the activity dependence of EpiPro: initial electrophysiology studies found that EpiPro-driven GFP expression was associated with increased firing rates when compared with unlabeled neurons, and the assessment of EpiPro-driven GFP expression revealed that GFP expression was increased ~150× after status epilepticus. Following this, we compared EpiPro-driven GFP expression in two rodent models of epilepsy, rat lithium/pilocarpine and mouse electrical kindling. In rodents with chronic epilepsy, GFP expression was increased in most neurons, but particularly in dentate granule cells, providing in vivo evidence to support the "breakdown of the dentate gate" hypothesis of limbic epileptogenesis. Finally, we assessed the time course of EpiPro activation and found that it was rapidly induced after seizures, with inactivation following over weeks, confirming EpiPro's potential utility as a gene therapy driver for epilepsy.

Drug-Inducible Gene Therapy Effectively Reduces Spontaneous Seizures in Kindled Rats but Creates Off-Target Side Effects in Inhibitory Neurons.

Over a third of patients with temporal lobe epilepsy (TLE) are not effectively treated with current anti-seizure drugs, spurring the development of gene therapies. The injection of adeno-associated viral vectors (AAV) into the brain has been shown to be a safe and viable approach. However, to date, AAV expression of therapeutic genes has not been regulated. Moreover, a common property of antiepileptic drugs is a narrow therapeutic window between seizure control and side effects. Therefore, a long-term goal is to develop drug-inducible gene therapies that can be regulated by clinically relevant drugs. In this study, a first-generation doxycycline-regulated gene therapy that delivered an engineered version of the leak potassium channel Kcnk2 (TREK-M) was injected into the hippocampus of male rats. Rats were electrically stimulated until kindled. EEG was monitored 24/7. Electrical kindling revealed an important side effect, as even low expression of TREK M in the absence of doxycycline was sufficient to cause rats to develop spontaneous recurring seizures. Treating the epileptic rats with doxycycline successfully reduced spontaneous seizures. Localization studies of infected neurons suggest seizures were caused by expression in GABAergic inhibitory neurons. In contrast, doxycycline increased the expression of TREK-M in excitatory neurons, thereby reducing seizures through net inhibition of firing. These studies demonstrate that drug-inducible gene therapies are effective in reducing spontaneous seizures and highlight the importance of testing for side effects with pro-epileptic stressors such as electrical kindling. These studies also show the importance of evaluating the location and spread of AAV-based gene therapies in preclinical studies.

Microglia play beneficial roles in multiple experimental seizure models.

Seizure disorders are common, affecting both the young and the old. Currently available antiseizure drugs are ineffective in a third of patients and have been developed with a focus on known neurocentric mechanisms, raising the need for investigations into alternative and complementary mechanisms that contribute to seizure generation or its containment. Neuroinflammation, broadly defined as the activation of immune cells and molecules in the central nervous system (CNS), has been proposed to facilitate seizure generation, although the specific cells involved in these processes remain inadequately understood. The role of microglia, the primary inflammation-competent cells of the brain, is debated since previous studies were conducted using approaches that were less specific to microglia or had inherent confounds. Using a selective approach to target microglia without such side effects, we show a broadly beneficial role for microglia in limiting chemoconvulsive, electrical, and hyperthermic seizures and argue for a further understanding of microglial contributions to contain seizures.

Microglia play beneficial roles in multiple experimental seizure models.

Seizure disorders are common, affecting both the young and the old. Currently available antiseizure drugs are ineffective in a third of patients and have been developed with a focus on known neurocentric mechanisms, raising the need for investigations into alternative and complementary mechanisms that contribute to seizure generation or its containment. Neuroinflammation, broadly defined as the activation of immune cells and molecules in the central nervous system (CNS), has been proposed to facilitate seizure generation, although the specific cells involved in these processes remain inadequately understood. The role of microglia, the primary inflammation-competent cells of the brain, is debated since previous studies were conducted using approaches that were less specific to microglia or had inherent confounds. Using a selective approach to target microglia without such side effects, we show a broadly beneficial role for microglia in limiting chemoconvulsive, electrical, and hyperthermic seizures and argue for a further understanding of microglial contributions to contain seizures.

Targeting oxidized phospholipids by AAV-based gene therapy in mice with established hepatic steatosis prevents progression to fibrosis.

Oxidized phosphatidylcholines (OxPCs) are implicated in chronic tissue damage. Hyperlipidemic LDL-R--deficient mice transgenic for an OxPC-recognizing IgM fragment (scFv-E06) are protected against nonalcoholic fatty liver disease (NAFLD). To examine the effect of OxPC elimination at different stages of NAFLD progression, we used cre-dependent, adeno-associated virus serotype 8-mediated expression of the single-chain variable fragment of E06 (AAV8-scFv-E06) in hepatocytes of albumin-cre mice. AAV8-induced expression of scFv-E06 at the start of FPC diet protected mice from developing hepatic steatosis. Independently, expression of scFv-E06 in mice with established steatosis prevented the progression to hepatic fibrosis. Mass spectrometry-based oxophospho-lipidomics identified individual OxPC species that were reduced by scFv-E06 expression. In vitro, identified OxPC species dysregulated mitochondrial metabolism and gene expression in hepatocytes and hepatic stellate cells. We demonstrate that individual OxPC species independently affect disease initiation and progression from hepatic steatosis to steatohepatitis, and that AAV-mediated expression of scFv-E06 is an effective therapeutic intervention.

AMPK-mediated potentiation of GABAergic signalling drives hypoglycaemia-provoked spike-wave seizures.

Metabolism regulates neuronal activity and modulates the occurrence of epileptic seizures. Here, using two rodent models of absence epilepsy, we show that hypoglycaemia increases the occurrence of spike-wave seizures. We then show that selectively disrupting glycolysis in the thalamus, a structure implicated in absence epilepsy, is sufficient to increase spike-wave seizures. We propose that activation of thalamic AMP-activated protein kinase, a sensor of cellular energetic stress and potentiator of metabotropic GABAB-receptor function, is a significant driver of hypoglycaemia-induced spike-wave seizures. We show that AMP-activated protein kinase augments postsynaptic GABAB-receptor-mediated currents in thalamocortical neurons and strengthens epileptiform network activity evoked in thalamic brain slices. Selective thalamic AMP-activated protein kinase activation also increases spike-wave seizures. Finally, systemic administration of metformin, an AMP-activated protein kinase agonist and common diabetes treatment, profoundly increased spike-wave seizures. These results advance the decades-old observation that glucose metabolism regulates thalamocortical circuit excitability by demonstrating that AMP-activated protein kinase and GABAB-receptor cooperativity is sufficient to provoke spike-wave seizures.

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Ion channels.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Effect of photobiomodulation on mitochondrial dynamics in peripheral nervous system in streptozotocin-induced type 1 diabetes in rats.

There is no effective treatment to halt peripheral nervous system damage in diabetic peripheral neuropathy. Mitochondria have been at the center of discussions as important factors in the development of neuropathy in diabetes. Photobiomodulation has been gaining clinical acceptance as it shows beneficial effects on a variety of nervous system disorders. In this study, the effects of photobiomodulation (904 nm, 45 mW, 6.23 J/cm2, 0.13 cm2, 60 ns pulsed time) on mitochondrial dynamics were evaluated in an adult male rat experimental model of streptozotocin-induced type 1 diabetes. Results presented here indicate that photobiomodulation could have an important role in preventing or reversing mitochondrial dynamics dysfunction in the course of peripheral nervous system damage in diabetic peripheral neuropathy. Photobiomodulation showed its effects on modulating the protein expression of mitofusin 2 and dynamin-related protein 1 in the sciatic nerve and in the dorsal root ganglia neurons of streptozotocin-induced type 1 diabetes in rats.

A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity1. The neural systems that regulate breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

Characterization of kindled VGAT-Cre mice as a new animal model of temporal lobe epilepsy.

OBJECTIVE: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism. METHODS: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology. RESULTS: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus. SIGNIFICANCE: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics.

Primary aldosteronism associated with a germline variant in CACNA1H.

The CACNA1H gene encodes the pore-forming α1 subunit of the T-type voltage-dependent calcium channel CaV3.2, expressed abundantly in the adrenal cortex. Mutations in CACNA1H are associated with various forms of primary aldosteronism (PA), including familial hyperaldosteronism type 4 (FH4). We describe a patient with refractory hypokalaemia and elevated aldosterone secretion independent of renin activity. Despite the absence of overt hypertension in this patient, the laboratory evaluation was consistent with a diagnosis of PA. Whole-exome sequencing revealed a de novo missense variant, R890H, in the voltage sensing domain of CACNA1H Expression of the variant channel in cells resulted in decreased whole-cell current, consistent with a loss-of-function. We hypothesise this variant is the genetic cause of pathological aldosterone secretion in this patient, and thereby expand the current understanding of the genetic basis of FH4.

Inhibition of T-Type calcium channels in mEC layer II stellate neurons reduces neuronal hyperexcitability associated with epilepsy.

Temporal lobe epilepsy (TLE) is a form of adult epilepsy involving the entorhinal cortex (EC). Layer II neurons of the medial EC (mEC) are spared and become hyperexcitable in TLE. Studies have suggested a role for T-type calcium channels (T-type Ca2+ channels) in facilitating increases in neuronal activity associated with TLE within the hippocampus. We sought to determine if T-type Ca2+ channels play a role in facilitating neuronal hyperexcitability of layer II mEC stellate neurons in TLE. TLE was induced in rats by electrical stimulation of the hippocampus to induce status epilepticus (SE). Brain slices were prepared from rats exhibiting spontaneous seizures and compared with age-matched control rats. Action potentials (APs) were evoked either by current injection steps or via presynaptic stimulation of mEC deep layers. The selective T-type Ca2+ channel antagonist, TTA-P2 (1 µM), was applied to determine the role of T-type Ca2+ channels in maintaining neuronal excitability. Quantitative PCR techniques were used to assess T-type Ca2+ channel isoform mRNA levels within the mEC layer II. TLE mEC layer II stellate neurons were hyperexcitable compared to control neurons, evoking a higher frequency of APs and generating bursts of APs when synaptically stimulated. TTA-P2 (1 µM) reduced firing frequencies in TLE and control neurons and reduced AP burst firing in TLE stellate neurons. TTA-P2 had little effect on synaptically evoked AP's in control neurons. TTA-P2 also inhibited rebound APs evoked in TLE neurons to a greater degree than in control neurons. TLE tissue had almost a 3-fold increase in Cav3.1 mRNA compared to controls. Cav3.2 or Cav3.3 levels were unchanged. These findings support a role for T-type Ca2+ channel in establishing neuronal hyperexcitability of mEC layer II stellate neurons in TLE. Increased expression of Cav3.1 may be important for establishing neuronal hyperexcitability of mEC layer II neurons in TLE.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

A novel therapeutic approach for treatment of catamenial epilepsy.

Many women with epilepsy experience perimenstrual seizure exacerbation, referred to as catamenial epilepsy. There is no effective treatment for this condition, proposed to result from withdrawal of neurosteroid-mediated effects of progesterone. A double-blind, multicenter, phase III, clinical trial of catamenial epilepsy has failed to find a beneficial effect of progesterone. The neurosteroid-mediated effects of progesterone have been extensively studied in relation to catamenial epilepsy; however, the effects mediated by progesterone receptor activation have been overlooked. We determined whether progesterone increased excitatory transmission in the hippocampus via activation of progesterone receptors, which may play a role in regulating catamenial seizure exacerbation. In a double-blind study using a rat model of catamenial epilepsy, we found that treatment with RU-486, which blocks progesterone and glucocorticoid receptors, significantly attenuated neurosteroid withdrawal-induced seizures. Furthermore, progesterone treatment as well as endogenous rise in progesterone during estrous cycle increased the expression of GluA1 and GluA2 subunits of AMPA receptors in the hippocampi, and enhanced the AMPA receptor-mediated synaptic transmission of CA1 pyramidal neurons. The progesterone-induced plasticity of AMPA receptors was blocked by RU-486 treatment and progesterone also failed to increase AMPA receptor expression in progesterone receptor knockout mice. These studies demonstrate that progesterone receptor activation regulates AMPA receptor expression and may play a role in catamenial seizure exacerbation.

Calmodulin regulates Cav3 T-type channels at their gating brake.

Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I-II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM.

Ca2+ Regulation of Cav3.3 T-type Ca2+ Channel Is Mediated by Calmodulin.

Calcium-dependent inactivation of high voltage-activated Ca2+ channels plays a crucial role in limiting rises in intracellular calcium (Ca2+i). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming α subunit. In contrast, little is known about how Ca2+i can regulate low voltage-activated T-type Ca2+ channels. Using whole cell patch clamp, we examined the biophysical properties of Ca2+ current through the three T-type Ca2+ channel isoforms, Cav3.1, Cav3.2, or Cav3.3, comparing internal solutions containing 27 nM and l µM free Ca2+ Both activation and inactivation kinetics of Cav3.3 current in l µM Ca2+i solution were more rapid than those in 27 nM Ca2+i solution. In addition, both activation and steady-state inactivation curves of Cav3.3 were negatively shifted in the higher Ca2+i solution. In contrast, the biophysical properties of Cav3.1 and Cav3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM1234 (a calmodulin mutant that doesn't bind Ca2+) occluded the effects of l µM Ca2+i on Cav3.3, implying that CaM is involved in the Ca2+i regulation effects on Cav3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Cav3.3. Taken together, our results suggest that Cav3.3 T-type channel is potently regulated by Ca2+i via interaction of Ca2+/CaM with the carboxyl terminus of Cav3.3.

Functional variants in HCN4 and CACNA1H may contribute to genetic generalized epilepsy.

Objective: Genetic generalized epilepsy (GGE) encompasses seizure disorders characterized by spike-and-wave discharges (SWD) originating within thalamo-cortical circuits. Hyperpolarization-activated (HCN) and T-type Ca2+ channels are key modulators of rhythmic activity in these brain regions. Here, we screened HCN4 and CACNA1H genes for potentially contributory variants and provide their functional analysis. Methods: Targeted gene sequencing was performed in 20 unrelated familial cases with different subtypes of GGE, and the results confirmed in 230 ethnically matching controls. Selected variants in CACNA1H and HCN4 were functionally assessed in tsA201 cells and Xenopus laevis oocytes, respectively. Results: We discovered a novel CACNA1H (p.G1158S) variant in two affected members of a single family. One of them also carried an HCN4 (p.P1117L) variant inherited from the unaffected mother. In a separate family, an HCN4 variant (p.E153G) was identified in one of several affected members. Voltage-clamp analysis of CACNA1H (p.G1158S) revealed a small but significant gain-of-function, including increased current density and a depolarizing shift of steady-state inactivation. HCN4 p.P1117L and p.G153E both caused a hyperpolarizing shift in activation and reduced current amplitudes, resulting in a loss-of-function. Significance: Our results are consistent with a model suggesting cumulative contributions of subtle functional variations in ion channels to seizure susceptibility and GGE.

Nalcn Is a "Leak" Sodium Channel That Regulates Excitability of Brainstem Chemosensory Neurons and Breathing.

UNLABELLED: The activity of background potassium and sodium channels determines neuronal excitability, but physiological roles for "leak" Na(+) channels in specific mammalian neurons have not been established. Here, we show that a leak Na(+) channel, Nalcn, is expressed in the CO2/H(+)-sensitive neurons of the mouse retrotrapezoid nucleus (RTN) that regulate breathing. In RTN neurons, Nalcn expression correlated with higher action potential discharge over a more alkalized range of activity; shRNA-mediated depletion of Nalcn hyperpolarized RTN neurons, and reduced leak Na(+) current and firing rate. Nalcn depletion also decreased RTN neuron activation by the neuropeptide, substance P, without affecting pH-sensitive background K(+) currents or activation by a cotransmitter, serotonin. In vivo, RTN-specific knockdown of Nalcn reduced CO2-evoked neuronal activation and breathing; hypoxic hyperventilation was unchanged. Thus, Nalcn regulates RTN neuronal excitability and stimulation by CO2, independent of direct pH sensing, potentially contributing to respiratory effects of Nalcn mutations; transmitter modulation of Nalcn may underlie state-dependent changes in breathing and respiratory chemosensitivity. SIGNIFICANCE STATEMENT: Breathing is an essential, enduring rhythmic motor activity orchestrated by dedicated brainstem circuits that require tonic excitatory drive for their persistent function. A major source of drive is from a group of CO2/H(+)-sensitive neurons in the retrotrapezoid nucleus (RTN), whose ongoing activity is critical for breathing. The ionic mechanisms that support spontaneous activity of RTN neurons are unknown. We show here that Nalcn, a unique channel that generates "leak" sodium currents, regulates excitability and neuromodulation of RTN neurons and CO2-stimulated breathing. Thus, this work defines a specific function for this enigmatic channel in an important physiological context.