Mechanism of an animal toxin-antidote system.

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

Chronic intermittent hypoxia elicits distinct transcriptomic responses among neurons and oligodendrocytes within the brainstem of mice.

Chronic intermittent hypoxia (CIH) is a prevalent condition characterized by recurrent episodes of oxygen deprivation, linked to respiratory and neurological disorders. Prolonged CIH is known to have adverse effects, including endothelial dysfunction, chronic inflammation, oxidative stress, and impaired neuronal function. These factors can contribute to serious comorbidities, including metabolic disorders and cardiovascular diseases. To investigate the molecular impact of CIH, we examined male C57BL/6J mice exposed to CIH for 21 days, comparing with normoxic controls. We used single-nucleus RNA sequencing to comprehensively examine the transcriptomic impact of CIH on key cell classes within the brainstem, specifically excitatory neurons, inhibitory neurons, and oligodendrocytes. These cell classes regulate essential physiological functions, including autonomic tone, cardiovascular control, and respiration. Through analysis of 10,995 nuclei isolated from pontine-medullary tissue, we identified seven major cell classes, further subdivided into 24 clusters. Our findings among these cell classes, revealed significant differential gene expression, underscoring their distinct responses to CIH. Notably, neurons exhibited transcriptional dysregulation of genes associated with synaptic transmission, and structural remodeling. In addition, we found dysregulated genes encoding ion channels and inflammatory response. Concurrently, oligodendrocytes exhibited dysregulated genes associated with oxidative phosphorylation and oxidative stress. Utilizing CellChat network analysis, we uncovered CIH-dependent altered patterns of diffusible intercellular signaling. These insights offer a comprehensive transcriptomic cellular atlas of the pons-medulla and provide a fundamental resource for the analysis of molecular adaptations triggered by CIH.NEW & NOTEWORTHY This study on chronic intermittent hypoxia (CIH) from pons-medulla provides initial insights into the molecular effects on excitatory neurons, inhibitory neurons, and oligodendrocytes, highlighting our unbiased approach, in comparison with earlier studies focusing on single target genes. Our findings reveal that CIH affects cell classes distinctly, and the dysregulated genes in distinct cell classes are associated with synaptic transmission, ion channels, inflammation, oxidative stress, and intercellular signaling, advancing our understanding of CIH-induced molecular responses.

AAV-mediated interneuron-specific gene replacement for Dravet syndrome.

Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy of SCN1A , which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV human SCN1A gene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form of SCN1A to circumvent AAV packaging limitations and deliver SCN1A via a dual vector approach using cell class-specific enhancers. These constructs produced full-length Na V 1.1 protein and functional sodium channels in HEK293 cells and in brain cells in vivo . After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles of Scn1a, at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves of SCN1A , or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting of SCN1A conferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediated SCN1A gene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.

Human KCNQ5 de novo mutations underlie epilepsy and intellectual disability.

We identified six novel de novo human KCNQ5 variants in children with motor/language delay, intellectual disability (ID), and/or epilepsy by whole exome sequencing. These variants, comprising two nonsense and four missense alterations, were functionally characterized by electrophysiology in HEK293/CHO cells, together with four previously reported KCNQ5 missense variants (Lehman A, Thouta S, Mancini GM, Naidu S, van Slegtenhorst M, McWalter K, Person R, Mwenifumbo J, Salvarinova R; CAUSES Study; EPGEN Study; Guella I, McKenzie MB, Datta A, Connolly MB, Kalkhoran SM, Poburko D, Friedman JM, Farrer MJ, Demos M, Desai S, Claydon T. Am J Hum Genet 101: 65-74, 2017). Surprisingly, all eight missense variants resulted in gain of function (GOF) due to hyperpolarized voltage dependence of activation or slowed deactivation kinetics, whereas the two nonsense variants were confirmed to be loss of function (LOF). One severe GOF allele (P369T) was tested and found to extend a dominant GOF effect to heteromeric KCNQ5/3 channels. Clinical presentations were associated with altered KCNQ5 channel gating: milder presentations with LOF or smaller GOF shifts in voltage dependence [change in voltage at half-maximal conduction (ΔV50) = ∼-15 mV] and severe presentations with larger GOF shifts in voltage dependence (ΔV50 = ∼-30 mV). To examine LOF pathogenicity, two Kcnq5 LOF mouse lines were created with CRISPR/Cas9. Both lines exhibited handling- and thermal-induced seizures and abnormal cortical EEGs consistent with epileptiform activity. Our study thus provides evidence for in vivo KCNQ5 LOF pathogenicity and strengthens the contribution of both LOF and GOF mutations to global pediatric neurological impairment, including ID/epilepsy.NEW & NOTEWORTHY Six novel de novo human KCNQ5 variants were identified from children with neurodevelopmental delay, intellectual disability, and/or epilepsy. Expression of these variants along with four previously reported KCNQ5 variants from a similar cohort revealed GOF potassium channels, negatively shifted in V50 of activation and/or delayed deactivation kinetics. GOF is extended to KCNQ5/3 heteromeric channels, making these the predominant channels affected in heterozygous de novo patients. Kcnq5 LOF mice exhibited seizures, consistent with in vivo pathogenicity.

Neuronal mechanisms underlying opioid-induced respiratory depression: our current understanding.

Opioid-induced respiratory depression (OIRD) represents the primary cause of death associated with therapeutic and recreational opioid use. Within the United States, the rate of death from opioid abuse since the early 1990s has grown disproportionally, prompting the classification as a nationwide "epidemic." Since this time, we have begun to unravel many fundamental cellular and systems-level mechanisms associated with opioid-related death. However, factors such as individual vulnerability, neuromodulatory compensation, and redundancy of opioid effects across central and peripheral nervous systems have created a barrier to a concise, integrative view of OIRD. Within this review, we bring together multiple perspectives in the field of OIRD to create an overarching viewpoint of what we know, and where we view this essential topic of research going forward into the future.

Presynaptic Mechanisms and KCNQ Potassium Channels Modulate Opioid Depression of Respiratory Drive.

Opioid-induced respiratory depression (OIRD) is the major cause of death associated with opioid analgesics and drugs of abuse, but the underlying cellular and molecular mechanisms remain poorly understood. We investigated opioid action in vivo in unanesthetized mice and in in vitro medullary slices containing the preBötzinger Complex (preBötC), a locus critical for breathing and inspiratory rhythm generation. Although hypothesized as a primary mechanism, we found that mu-opioid receptor (MOR1)-mediated GIRK activation contributed only modestly to OIRD. Instead, mEPSC recordings from genetically identified Dbx1-derived interneurons, essential for rhythmogenesis, revealed a prevalent presynaptic mode of action for OIRD. Consistent with MOR1-mediated suppression of presynaptic release as a major component of OIRD, Cacna1a KO slices lacking P/Q-type Ca2+ channels enhanced OIRD. Furthermore, OIRD was mimicked and reversed by KCNQ potassium channel activators and blockers, respectively. In vivo whole-body plethysmography combined with systemic delivery of GIRK- and KCNQ-specific potassium channel drugs largely recapitulated these in vitro results, and revealed state-dependent modulation of OIRD. We propose that respiratory failure from OIRD results from a general reduction of synaptic efficacy, leading to a state-dependent collapse of rhythmic network activity.

Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans.

Adaptive responses to external temperatures are essential for survival in changing environments. We show here that environmental oxygen concentration affects cold acclimation in Caenorhabditis elegans and that this response is regulated by a KCNQ-type potassium channel, KQT-2. Depending on culture conditions, kqt-2 mutants showed supranormal cold acclimation, caused by abnormal thermosensation in ADL chemosensory neurons. ADL neurons are responsive to temperature via transient receptor potential channels-OSM-9, OCR-2, and OCR-1-with OCR-1 negatively regulating ADL function. Similarly, KQT-2 and KQT-3 regulate ADL activity, with KQT-2 positively regulating ADL function. Abnormal cold acclimation and acute temperature responses of ADL neurons in kqt-2 mutants were suppressed by an oxygen-receptor mutation in URX coelomic sensory neurons, which are electrically connected to ADL via RMG interneurons. Likewise, low oxygen suppressed supranormal kqt-2 cold acclimation. These data thus demonstrate a simple neuronal circuit integrating two different sensory modalities, temperature and oxygen, that determines cold acclimation.

International Union of Basic and Clinical Pharmacology. C. Nomenclature and Properties of Calcium-Activated and Sodium-Activated Potassium Channels.

A subset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not all members are sensitive to calcium. This article describes the molecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels.

A novel excitatory network for the control of breathing.

Breathing must be tightly coordinated with other behaviours such as vocalization, swallowing, and coughing. These behaviours occur after inspiration, during a respiratory phase termed postinspiration. Failure to coordinate postinspiration with inspiration can result in aspiration pneumonia, the leading cause of death in Alzheimer's disease, Parkinson's disease, dementia, and other neurodegenerative diseases. Here we describe an excitatory network that generates the neuronal correlate of postinspiratory activity in mice. Glutamatergic-cholinergic neurons form the basis of this network, and GABA (γ-aminobutyric acid)-mediated inhibition establishes the timing and coordination relative to inspiration. We refer to this network as the postinspiratory complex (PiCo). The PiCo has autonomous rhythm-generating properties and is necessary and sufficient for postinspiratory activity in vivo.The PiCo also shows distinct responses to neuromodulators when compared to other excitatory brainstem networks. On the basis of the discovery of the PiCo, we propose that each of the three phases of breathing is generated by a distinct excitatory network: the pre-Bötzinger complex, which has been linked to inspiration; the PiCo, as described here for the neuronal control of postinspiration; and the lateral parafacial region (pF(L)), which has been associated with active expiration, a respiratory phase that is recruited during high metabolic demand.

When norepinephrine becomes a driver of breathing irregularities: how intermittent hypoxia fundamentally alters the modulatory response of the respiratory network.

Neuronal networks are endogenously modulated by aminergic and peptidergic substances. These modulatory processes are critical for maintaining normal activity and adapting networks to changes in metabolic, behavioral, and environmental conditions. However, disturbances in neuromodulation have also been associated with pathologies. Using whole animals (in vivo) and functional brainstem slices (in vitro) from mice, we demonstrate that exposure to acute intermittent hypoxia (AIH) leads to fundamental changes in the neuromodulatory response of the respiratory network located within the preBötzinger complex (preBötC), an area critical for breathing. Norepinephrine, which normally regularizes respiratory activity, renders respiratory activity irregular after AIH. Respiratory irregularities are caused both in vitro and in vivo by AIH, which increases synaptic inhibition within the preBötC when norepinephrine is endogenously or exogenously increased. These irregularities are prevented by blocking synaptic inhibition before AIH. However, regular breathing cannot be reestablished if synaptic inhibition is blocked after AIH. We conclude that subtle changes in synaptic transmission can have dramatic consequences at the network level as endogenously released neuromodulators that are normally adaptive become the drivers of irregularity. Moreover, irregularities in the preBötC result in irregularities in the motor output in vivo and in incomplete transmission of inspiratory activity to the hypoglossus motor nucleus. Our finding has basic science implications for understanding network functions in general, and it may be clinically relevant for understanding pathological disturbances associated with hypoxic episodes such as those associated with myocardial infarcts, obstructive sleep apneas, apneas of prematurity, Rett syndrome, and sudden infant death syndrome.

Medial habenula output circuit mediated by α5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus.

The Chrna5 gene encodes the α5 nicotinic acetylcholine receptor subunit, an "accessory" subunit of pentameric nicotinic receptors, that has been shown to play a role in nicotine-related behaviors in rodents and is genetically linked to smoking behavior in humans. Here we have used a BAC transgenic mouse line, α5(GFP), to examine the cellular phenotype, connectivity, and function of α5-expressing neurons. Although the medial habenula (MHb) has been proposed as a site of α5 function, α5(GFP) is not detectable in the MHb, and α5 mRNA is expressed there only at very low levels. However, α5(GFP) is strongly expressed in a subset of neurons in the interpeduncular nucleus (IP), median raphe/paramedian raphe (MnR/PMnR), and dorsal tegmental area (DTg). Double-label fluorescence in situ hybridization reveals that these neurons are exclusively GABAergic. Transgenic and conventional tract tracing show that α5(GFP) neurons in the IP project principally to the MnR/PMnR and DTg/interfascicular dorsal raphe, both areas rich in serotonergic neurons. The α5(GFP) neurons in the IP are located in a region that receives cholinergic fiber inputs from the ventral MHb, and optogenetically assisted circuit mapping demonstrates a monosynaptic connection between these cholinergic neurons and α5(GFP) IP neurons. Selective inhibitors of both α4ß2- and α3ß4-containing nicotinic receptors were able to reduce nicotine-evoked inward currents in α5(GFP) neurons in the IP, suggesting a mixed nicotinic receptor profile in these cells. Together, these findings show that the α5-GABAergic interneurons form a link from the MHb to serotonergic brain centers, which is likely to mediate some of the behavioral effects of nicotine.

Inter-α/ß subunits coupling mediating pre-inactivation and augmented activation of BKCa(ß2).

Large-conductance calcium-activated potassium (BK) channels regulate the electric properties and neurotransmitter release in excitable cells. Its auxiliary ß2 subunits not only enhance gating, but also confer inactivation via a short-lived preinactivated state. However, the mechanism of enhancement and preinactivation of BK channels by ß2 remains elusive. Using our newly developed methods, we demonstrated that electrostatic forces played a crucial role in forming multiple complementary pairs of binding sites between α and ß subunits including a "PI site" required for channel preinactivation, an "E site" enhancing calcium sensitivity and an "ECaB" coupling site transferring force to gate from the Ca(2+)-bowl via the ß2(K33, R34, K35), E site and S6-C linker, independent of another Ca(2+) binding site mSlo1(D362,D367). A comprehensive structural model of the BK(ß2) complex was reconstructed based on these functional studies, which paves the way for a clearer understanding of the structural mechanisms of activation and preinactivation of other BK(ß) complexes.

Stable respiratory activity requires both P/Q-type and N-type voltage-gated calcium channels.

P/Q-type voltage-gated calcium channels (Ca(v)2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Ca(v)2.1 pore-forming α(1A) subunit (α(1A)⁻/⁻) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolated in vitro brainstem slice preparation containing the pre-Bötzinger complex. The loss of Ca(v)2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Ca(v)2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α(1A)⁻/⁻ mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α(1A)⁻/⁻ mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α(1A)⁻/⁻ mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α(1A)⁻/⁻ mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.

Chromanol 293B binding in KCNQ1 (Kv7.1) channels involves electrostatic interactions with a potassium ion in the selectivity filter.

The chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chroman) is a lead compound of potential class III antiarrhythmics that inhibit cardiac I(Ks) potassium channels. These channels are formed by the coassembly of KCNQ1 (Kv7.1, KvLQT1) and KCNE1 subunits. Although homomeric KCNQ1 channels are the principal molecular targets, entry of KCNE1 to the channel complex enhances the chromanol block. Because closely related neuronal KCNQ2 potassium channels are insensitive to the drug, we used KCNQ1/KCNQ2 chimeras to identify the binding site of the inhibitor. We localized the putative drug receptor to the H5 selectivity filter and the S6 transmembrane segment. Single residues affecting 293B inhibition were subsequently identified through systematic exchange of amino acids that were either different in KCNQ1 and KCNQ2 or predicted by a docking model of 293B in the open and closed conformation of KCNQ1. Mutant channel proteins T312S, I337V, and F340Y displayed dramatically lowered sensitivity to chromanol block. The predicted drug binding receptor lies in the inner pore vestibule containing the lower part of the selectivity filter, and the S6 transmembrane domain also reported to be important for binding of benzodiazepines. We propose that the block of the ion permeation pathway involves hydrophobic interactions with the S6 transmembrane residues Ile337 and Phe340, and stabilization of chromanol 293B binding through electrostatic interactions of its oxygen atoms with the most internal potassium ion within the selectivity filter.

KCNQ-like potassium channels in Caenorhabditis elegans. Conserved properties and modulation.

The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C.