New insights into the physiology and pathophysiology of the atypical sodium leak channel NALCN.

Cell excitability and its modulation by hormones and neurotransmitters involve the concerted action of a large repertoire of membrane proteins, especially ion channels. Unique complements of coexpressed ion channels are exquisitely balanced against each other in different excitable cell types, establishing distinct electrical properties that are tailored for diverse physiological contributions, and dysfunction of any component may induce a disease state. A crucial parameter controlling cell excitability is the resting membrane potential (RMP) set by extra- and intracellular concentrations of ions, mainly Na+, K+, and Cl-, and their passive permeation across the cell membrane through leak ion channels. Indeed, dysregulation of RMP causes significant effects on cellular excitability. This review describes the molecular and physiological properties of the Na+ leak channel NALCN, which associates with its accessory subunits UNC-79, UNC-80, and NLF-1/FAM155 to conduct depolarizing background Na+ currents in various excitable cell types, especially neurons. Studies of animal models clearly demonstrate that NALCN contributes to fundamental physiological processes in the nervous system including the control of respiratory rhythm, circadian rhythm, sleep, and locomotor behavior. Furthermore, dysfunction of NALCN and its subunits is associated with severe pathological states in humans. The critical involvement of NALCN in physiology is now well established, but its study has been hampered by the lack of specific drugs that can block or agonize NALCN currents in vitro and in vivo. Molecular tools and animal models are now available to accelerate our understanding of how NALCN contributes to key physiological functions and the development of novel therapies for NALCN channelopathies.

NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Sodium background currents in endocrine/neuroendocrine cells: Towards unraveling channel identity and contribution in hormone secretion.

In endocrine/neuroendocrine tissues, excitability of secretory cells is patterned by the repertoire of ion channels and there is clear evidence that extracellular sodium (Na+) ions contribute to hormone secretion. While voltage-gated channels involved in action potential generation are well-described, the background 'leak' channels operating near the resting membrane potential are much less known, and in particular the channels supporting a background entry of Na+ ions. These background Na+ currents (called here 'INab') have the ability to modulate the resting membrane potential and subsequently affect action potential firing. Here we compile and analyze the data collected from three endocrine/neuroendocrine tissues: the anterior pituitary gland, the adrenal medulla and the endocrine pancreas. We also model how INab can be functionally involved in cellular excitability. Finally, towards deciphering the physiological role of INab in endocrine/neuroendocrine cells, its implication in hormone release is also discussed.

The sodium leak channel NALCN regulates cell excitability of pituitary endocrine cells.

Anterior pituitary endocrine cells that release hormones such as growth hormone and prolactin are excitable and fire action potentials. In these cells, several studies previously showed that extracellular sodium (Na+ ) removal resulted in a negative shift of the resting membrane potential (RMP) and a subsequent inhibition of the spontaneous firing of action potentials, suggesting the contribution of a Na+ background conductance. Here, we show that the Na+ leak channel NALCN conducts a Ca2+ - Gd3+ -sensitive and TTX-resistant Na+ background conductance in the GH3 cell line, a cell model of pituitary endocrine cells. NALCN knockdown hyperpolarized the RMP, altered GH3 cell electrical properties and inhibited prolactin secretion. Conversely, the overexpression of NALCN depolarized the RMP, also reshaping the electrical properties of GH3 cells. Overall, our results indicate that NALCN is functional in GH3 cells and involved in endocrine cell excitability as well as in hormone secretion. Indeed, the GH3 cell line suitably models native pituitary cells that display a similar Na+ background conductance and appears as a proper cellular model to study the role of NALCN in cellular excitability.

A sodium background conductance controls the spiking pattern of mouse adrenal chromaffin cells in situ.

KEY POINTS: Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode. A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+ -containing extracellular saline. This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I-V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN. In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells. The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus-secretion coupling of the adrenomedullary tissue. ABSTRACT: Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely-tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na+ -containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I-V relationship between -110 and -40 mV, and (iii) is carried by cations with a conductance sequence gNa  > gK  > gCs . These biophysical attributes, together with the expression of the sodium-leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus-secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands.

Neuronal Cav3 channelopathies: recent progress and perspectives.

T-type, low-voltage activated, calcium channels, now designated Cav3 channels, are involved in a wide variety of physiological functions, especially in nervous systems. Their unique electrophysiological properties allow them to finely regulate neuronal excitability and to contribute to sensory processing, sleep, and hormone and neurotransmitter release. In the last two decades, genetic studies, including exploration of knock-out mouse models, have greatly contributed to elucidate the role of Cav3 channels in normal physiology, their regulation, and their implication in diseases. Mutations in genes encoding Cav3 channels (CACNA1G, CACNA1H, and CACNA1I) have been linked to a variety of neurodevelopmental, neurological, and psychiatric diseases designated here as neuronal Cav3 channelopathies. In this review, we describe and discuss the clinical findings and supporting in vitro and in vivo studies of the mutant channels, with a focus on de novo, gain-of-function missense mutations recently discovered in CACNA1G and CACNA1H. Overall, the studies of the Cav3 channelopathies help deciphering the pathogenic mechanisms of corresponding diseases and better delineate the properties and physiological roles Cav3 channels.

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.

A Recurrent Mutation in CACNA1G Alters Cav3.1 T-Type Calcium-Channel Conduction and Causes Autosomal-Dominant Cerebellar Ataxia.

Hereditary cerebellar ataxias (CAs) are neurodegenerative disorders clinically characterized by a cerebellar syndrome, often accompanied by other neurological or non-neurological signs. All transmission modes have been described. In autosomal-dominant CA (ADCA), mutations in more than 30 genes are implicated, but the molecular diagnosis remains unknown in about 40% of cases. Implication of ion channels has long been an ongoing topic in the genetics of CA, and mutations in several channel genes have been recently connected to ADCA. In a large family affected by ADCA and mild pyramidal signs, we searched for the causative variant by combining linkage analysis and whole-exome sequencing. In CACNA1G, we identified a c.5144G>A mutation, causing an arginine-to-histidine (p.Arg1715His) change in the voltage sensor S4 segment of the T-type channel protein Cav3.1. Two out of 479 index subjects screened subsequently harbored the same mutation. We performed electrophysiological experiments in HEK293T cells to compare the properties of the p.Arg1715His and wild-type Cav3.1 channels. The current-voltage and the steady-state activation curves of the p.Arg1715His channel were shifted positively, whereas the inactivation curve had a higher slope factor. Computer modeling in deep cerebellar nuclei (DCN) neurons suggested that the mutation results in decreased neuronal excitability. Taken together, these data establish CACNA1G, which is highly expressed in the cerebellum, as a gene whose mutations can cause ADCA. This is consistent with the neuropathological examination, which showed severe Purkinje cell loss. Our study further extends our knowledge of the link between calcium channelopathies and CAs.

De novo mutations in NALCN cause a syndrome characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop.

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser(423)-Pro(542)) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels.

Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2- and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis.

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

Emerging and re-emerging zoonotic viral diseases in Southeast Asia: One Health challenge.

The ongoing significant social, environmental, and economic changes in Southeast Asia (SEA) make the region highly vulnerable to the emergence and re-emergence of zoonotic viral diseases. In the last century, SEA has faced major viral outbreaks with great health and economic impact, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), arboviruses, highly pathogenic avian influenza (H5N1), and Severe Acute Respiratory Syndrome (SARS-CoV); and so far, imported cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Given the recent challenging experiences in addressing emerging zoonotic diseases, it is necessary to redouble efforts to effectively implement the "One Health" initiative in the region, which aims to strengthen the human-animal-plant-environment interface to better prevent, detect and respond to health threats while promoting sustainable development. This review provides an overview of important emerging and re-emerging zoonotic viral diseases in SEA, with emphasis on the main drivers behind their emergency, the epidemiological situation from January 2000 to October 2022, and the importance of One Health to promote improved intervention strategies.

Functional mapping of microRNA promoters with dCas9 fused to transcriptional regulators.

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types.

Effects of Ceftriaxone on Oxidative Stress and Inflammation in a Rat Model of Chronic Cerebral Hypoperfusion.

Ceftriaxone (CTX) exerts a neuroprotective effect by decreasing glutamate excitotoxicity. We further studied the underlying mechanisms and effects of CTX early post-treatment on behavior in a cerebral hypoperfusion rats. The rats' common carotid arteries (2VO) were permanently ligated. CTX was treated after ischemia. Biochemical studies were performed to assess antioxidative stress and inflammation. Behavioral and histological studies were then tested on the ninth week after vessel ligation. The 2VO rats showed learning and memory deficits as well as working memory impairments without any motor weakness. The treatment with CTX was found to attenuate white matter damage, MDA production, and interleukin 1 beta and tumor necrosis factor alpha production, mainly in the hippocampal area. Moreover, CTX treatment could increase the expression of glia and the glial glutamate transporters, and the neuronal glutamate transporter. Taken together, our data indicate the neuroprotective mechanisms of CTX involving the upregulation of glutamate transporters' expression. This increased expression contributes to a reduction in glutamate excitotoxicity and oxidative stress as well as pro-inflammatory cytokine production, thus resulting in the protection of neurons and tissue from further damage. The present study highlights the mechanism of the effect of CTX treatment and of the underlying ischemia-induced neuronal damage.

The sodium "leak" has finally been plugged.

Most electrophysiologists generally do not speak highly of leak currents. In reality, these conductances represent a crucial functional mechanism by which neurons control resting membrane potentials. A new study in Cell by Lu et al. has surprisingly confirmed the identity of the long-sought voltage-insensitive sodium leak conductance to be encoded by the third branch of the voltage-gated sodium and calcium channel family.

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic beta-cell line.

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic beta-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I-II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.

The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells.

The SCN4B gene, coding for the NaVß4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVß4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVß4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of ß-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navß4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.

Regulation of T-type calcium channels: signalling pathways and functional implications.

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.

A destructive interaction mechanism accounts for dominant-negative effects of misfolded mutants of voltage-gated calcium channels.

Channelopathies are often linked to defective protein folding and trafficking. Among them, the calcium channelopathy episodic ataxia type-2 (EA2) is an autosomal dominant disorder related to mutations in the pore-forming Ca(v)2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca(v)2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (D(I-II)) of the Ca(v)3.2 subunit of T-type calcium channel are misfolded, retained in the endoplasmic reticulum, and subject to proteasomal degradation. Pulse-chase experiments revealed that misfolded mutants bind to nascent wild-type Ca(v) subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca(v) mutants is likely to occur in EA2 and in other inherited dominant channelopathies.