Repertoire Of Channels Research Articles

Characterization of ClC-1 chloride channels in zebrafish: a new model to study myotonia.

The function of the chloride channel ClC-1 is crucial for the control of muscle excitability. Thus, reduction of ClC-1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc-1a and clc-1b) of the ClC-1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT-PCR, western blot, and electrophysiological recordings of myotubes, and clc-1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9-anthracenecarboxylic acid (9-AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC-1 but not by some ClC-1 containing myotonia mutations. Treatment of clc-1-depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure-function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. KEY POINTS: We have identified two orthologues of ClC-1 in zebrafish (clc-1a and clc-1b) which are mostly expressed in skeletal muscle at different developmental stages. Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9-AC and acidic pH inverts their voltage dependence of gating. Reduction of clc-1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC-1. Myotonia-like symptoms caused by clc-1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia.

Evolution of ion channels in cetaceans: a natural experiment in the tree of life

Cetaceans represent a natural experiment within the tree of life in which a lineage changed from terrestrial to aquatic habitats. This shift involved phenotypic modifications, representing an opportunity to explore the genetic bases of phenotypic diversity. Among the different molecular systems that maintain cellular homeostasis, ion channels are crucial for the proper physiological functioning of all living species. This study aims to explore the evolution of ion channels during the evolutionary history of cetaceans. To do so, we created a bioinformatic pipeline to annotate the repertoire of ion channels in the genome of the species included in our sampling. Our main results show that cetaceans have, on average, fewer protein-coding genes and a higher percentage of annotated ion channels than non-cetacean mammals. Signals of positive selection were detected in ion channels related to the heart, locomotion, visual and neurological phenotypes. Interestingly, we predict that the NaV1.5 ion channel of most toothed whales (odontocetes) is sensitive to tetrodotoxin, similar to NaV1.7, given the presence of tyrosine instead of cysteine, in a specific position of the ion channel. Finally, the gene turnover rate of the cetacean crown group is more than three times faster than that of non-cetacean mammals.

Change of voltage-gated sodium channel repertoire in skeletal muscle of a MuSK myasthenia gravis mouse model.

Muscle-specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter-patient and inter-muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by μ-Conotoxin-GIIIB, a blocker of skeletal muscle NaV1.4 voltage-gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co-expression of (μ-Conotoxin-GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno-compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, μ-Conotoxin-GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High- and intermediate-dosed mice showed clear, progressive myasthenia, not seen in low-dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low-dosed mouse diaphragms showed a much higher degree of μ-Conotoxin-GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed μ-Conotoxin-GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect.

Spatially resolved multiomics of human cardiac niches

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug–target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Biological complexity facilitates tuning of the neuronal parameter space.

The electrical and computational properties of neurons in our brains are determined by a rich repertoire of membrane-spanning ion channels and elaborate dendritic trees. However, the precise reason for this inherent complexity remains unknown, given that simpler models with fewer ion channels are also able to functionally reproduce the behaviour of some neurons. Here, we stochastically varied the ion channel densities of a biophysically detailed dentate gyrus granule cell model to produce a large population of putative granule cells, comparing those with all 15 original ion channels to their reduced but functional counterparts containing only 5 ion channels. Strikingly, valid parameter combinations in the full models were dramatically more frequent at ~6% vs. ~1% in the simpler model. The full models were also more stable in the face of perturbations to channel expression levels. Scaling up the numbers of ion channels artificially in the reduced models recovered these advantages confirming the key contribution of the actual number of ion channel types. We conclude that the diversity of ion channels gives a neuron greater flexibility and robustness to achieve a target excitability.

Motor cortex analogue neurons in songbirds utilize Kv3 channels to generate ultranarrow spikes.

Complex motor skills in vertebrates require specialized upper motor neurons with precise action potential (AP) firing. To examine how diverse populations of upper motor neurons subserve distinct functions and the specific repertoire of ion channels involved, we conducted a thorough study of the excitability of upper motor neurons controlling somatic motor function in the zebra finch. We found that robustus arcopallialis projection neurons (RAPNs), key command neurons for song production, exhibit ultranarrow spikes and higher firing rates compared to neurons controlling non-vocal somatic motor functions (dorsal intermediate arcopallium [AId] neurons). Pharmacological and molecular data indicate that this striking difference is associated with the higher expression in RAPNs of high threshold, fast-activating voltage-gated Kv3 channels, that likely contain Kv3.1 (KCNC1) subunits. The spike waveform and Kv3.1 expression in RAPNs mirror properties of Betz cells, specialized upper motor neurons involved in fine digit control in humans and other primates but absent in rodents. Our study thus provides evidence that songbirds and primates have convergently evolved the use of Kv3.1 to ensure precise, rapid AP firing in upper motor neurons controlling fast and complex motor skills.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

A Novel and Functionally Diverse Class of Acetylcholine-Gated Ion Channels.

Fast cholinergic neurotransmission is mediated by acetylcholine-gated ion channels; in particular, excitatory nicotinic acetylcholine receptors play well established roles in virtually all nervous systems. Acetylcholine-gated inhibitory channels have also been identified in some invertebrate phyla, yet their roles in the nervous system are less well understood. We report the existence of multiple new inhibitory ion channels with diverse ligand activation properties in Caenorhabditis elegans We identify three channels, LGC-40, LGC-57, and LGC-58, whose primary ligand is choline rather than acetylcholine, as well as the first evidence of a truly polymodal channel, LGC-39, which is activated by both cholinergic and aminergic ligands. Using our new ligand-receptor pairs we uncover the surprising extent to which single neurons in the hermaphrodite nervous system express both excitatory and inhibitory channels, not only for acetylcholine but also for the other major neurotransmitters. The results presented in this study offer new insight into the potential evolutionary benefit of a vast and diverse repertoire of ligand-gated ion channels to generate complexity in an anatomically compact nervous system.SIGNIFICANCE STATEMENT Here we describe the diversity of cholinergic signaling in the nematode Caenorhabditis elegans We identify and characterize a novel family of ligand-gated ion channels and show that they are preferentially gated by choline rather than acetylcholine and expressed broadly in the nervous system. Interestingly, we also identify one channel gated by chemically diverse ligands including acetylcholine and aminergic ligands. By using our new knowledge of these ligand-gated ion channels, we built a model to predict the synaptic polarity in the C. elegans connectome. This model can be used for generating hypotheses on neural circuit function.

Ancient multiplicity in cyclic nucleotide-gated (CNG) cation channel repertoire was reduced in the ancestor of Olfactores before re-expansion by whole genome duplications in vertebrates.

Cyclic nucleotide-gated (CNG) cation channels are important heterotetrameric proteins in the retina, with different subunit composition in cone and rod photoreceptor cells: three CNGA3 and one CNGB3 in cones and three CNGA1 and one CNGB1 in rods. CNGA and CNGB subunits form separate subfamilies. We have analyzed the evolution of the CNG gene family in metazoans, with special focus on vertebrates by using sequence-based phylogeny and conservation of chromosomal synteny to deduce paralogons resulting from the early vertebrate whole genome duplications (WGDs). Our analyses show, unexpectedly, that the CNGA subfamily had four sister subfamilies in the ancestor of bilaterians and cnidarians that we named CNGC, CNGD, CNGE and CNGF. Of these, CNGC, CNGE and CNGF were lost in the ancestor of Olfactores while CNGD was lost in the vertebrate ancestor. The remaining CNGA and CNGB genes were expanded by a local duplication of CNGA and the subsequent chromosome duplications in the basal vertebrate WGD events. Upon some losses, this resulted in the gnathostome ancestor having three members in the visual CNGA subfamily (CNGA1-3), a single CNGA4 gene, and two members in the CNGB subfamily (CNGB1 and CNGB3). The nature of chromosomal rearrangements in the vertebrate CNGA paralogon was resolved by including the genomes of a non-teleost actinopterygian and an elasmobranch. After the teleost-specific WGD, additional duplicates were generated and retained for CNGA1, CNGA2, CNGA3 and CNGB1. Furthermore, teleosts retain a local duplicate of CNGB3. The retention of duplicated CNG genes is explained by their subfunctionalisation and photoreceptor-specific expression. In conclusion, this study provides evidence for four previously unknown CNG subfamilies in metazoans and further evidence that the early vertebrate WGD events were instrumental in the evolution of the vertebrate visual and central nervous systems.

Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a Painless homolog

The transient receptor potential (TRP) family of cation channels are evolutionarily conserved proteins with critical roles in sensory physiology. Despite extensive studies in model species, knowledge of TRP channel functional diversity and physiological impact remains limited in many non-model insect species. To assess the TRP channel repertoire in a non-model agriculture pest species (Lygus hesperus), publicly available transcriptomic datasets were mined for potential homologs. Among the transcripts identified, 30 are predicted to encompass complete open reading frames that encode proteins representing each of the seven TRP channel subfamilies. Although no homologs were identified for the Pyrexia and Brivido channels, the TRP complement in L. hesperus exceeded the 13–16 channels reported in most insects. This diversity appears to be driven by a combination of alternative splicing, which impacted members of six subfamilies, and gene expansion of the TRPP subfamily. To validate the in silico data and provide more detailed analyses of L. hesperus TRP functionality, the putative Painless homolog was selected for more in depth analysis and its functional role in thermosensation examined in vitro. RT-PCR expression profiling revealed near ubiquitous expression of the Painless transcript throughout nymphal and adult development. Electrophysiological data generated using a Xenopus oocyte recombinant expression system indicated activation parameters for L. hesperus Painless homolog that are consistent with a role in noxious heat (40°–45 °C) thermosensation.

Inactivation of a New Potassium Channel Increases Rifampicin Resistance and Induces Collateral Sensitivity to Hydrophilic Antibiotics in Mycobacterium smegmatis

Rifampicin is a critical first-line antibiotic for treating mycobacterial infections such as tuberculosis, one of the most serious infectious diseases worldwide. Rifampicin resistance in mycobacteria is mainly caused by mutations in the rpoB gene; however, some rifampicin-resistant strains showed no rpoB mutations. Therefore, alternative mechanisms must explain this resistance in mycobacteria. In this work, a library of 11,000 Mycobacterium smegmatis mc2 155 insertion mutants was explored to search and characterize new rifampicin-resistance determinants. A transposon insertion in the MSMEG_1945 gene modified the growth rate, pH homeostasis and membrane potential in M. smegmatis, producing rifampicin resistance and collateral susceptibility to other antitubercular drugs such as isoniazid, ethionamide and aminoglycosides. Our data suggest that the M. smegmatis MSMEG_1945 protein is an ion channel, dubbed MchK, essential for maintaining the cellular ionic balance and membrane potential, modulating susceptibility to antimycobacterial agents. The functions of this new gene point once again to potassium homeostasis impairment as a proxy to resistance to rifampicin. This study increases the known repertoire of mycobacterial ion channels involved in drug susceptibility/resistance to antimycobacterial drugs and suggests novel intervention opportunities, highlighting ion channels as druggable pathways.

Ion channels and neuronal excitability in polyglutamine neurodegenerative diseases.

Polyglutamine (polyQ) diseases are a family composed of nine neurodegenerative inherited disorders (NDDs) caused by pathological expansions of cytosine-adenine-guanine (CAG) trinucleotide repeats which encode a polyQ tract in the corresponding proteins. CAG polyQ repeat expansions produce neurodegeneration via multiple downstream mechanisms; among those the neuronal activity underlying the ion channels is affected directly by specific channelopathies or indirectly by secondary dysregulation. In both cases, the altered excitability underlies to gain- or loss-of-function pathological effects. Here we summarize the repertoire of ion channels in polyQ NDDs emphasizing the biophysical features of neuronal excitability and their pathogenic role. The aim of this review is to point out the value of a deeper understanding of those functional mechanisms and processes as crucial elements for the designing and targeting of novel therapeutic avenues.

Astrocytic potassium and calcium channels as integrators of the inflammatory and ischemic CNS microenvironment.

Astrocytes are key regulators of their surroundings by receiving and integrating stimuli from their local microenvironment, thereby regulating glial and neuronal homeostasis. Cumulating evidence supports a plethora of heterogenic astrocyte subpopulations that differ morphologically and in their expression patterns of receptors, transporters and ion channels, as well as in their functional specialisation. Astrocytic heterogeneity is especially relevant under pathological conditions. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), morphologically distinct astrocytic subtypes were identified and could be linked to transcriptome changes during different disease stages and regions. To allow for continuous awareness of changing stimuli across age and diseases, astrocytes are equipped with a variety of receptors and ion channels allowing the precise perception of environmental cues. Recent studies implicate the diverse repertoire of astrocytic ion channels- including transient receptor potential channels, voltage-gated calcium channels, inwardly rectifying K+ channels, and two-pore domain potassium channels- in sensing the brain state in physiology, inflammation and ischemia. Here, we review current evidence regarding astrocytic potassium and calcium channels and their functional contribution in homeostasis, neuroinflammation and stroke.

Loss of HCN2 in Dorsal Hippocampus of Young Adult Mice Induces Specific Apoptosis of the CA1 Pyramidal Neuron Layer.

Neurons inevitably rely on a proper repertoire and distribution of membrane-bound ion-conducting channels. Among these proteins, the family of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels possesses unique properties giving rise to the corresponding Ih-current that contributes to various aspects of neural signaling. In mammals, four genes (hcn1-4) encode subunits of HCN channels. These subunits can assemble as hetero- or homotetrameric ion-conducting channels. In order to elaborate on the specific role of the HCN2 subunit in shaping electrical properties of neurons, we applied an Adeno-associated virus (AAV)-mediated, RNAi-based knock-down strategy of hcn2 gene expression both in vitro and in vivo. Electrophysiological measurements showed that HCN2 subunit knock-down resulted in specific yet anticipated changes in Ih-current properties in primary hippocampal neurons and, in addition, corroborated that the HCN2 subunit participates in postsynaptic signal integration. To further address the role of the HCN2 subunit in vivo, we injected recombinant (r)AAVs into the dorsal hippocampus of young adult male mice. Behavioral and biochemical analyses were conducted to assess the contribution of HCN2-containing channels in shaping hippocampal network properties. Surprisingly, knock-down of hcn2 expression resulted in a severe degeneration of the CA1 pyramidal cell layer, which did not occur in mice injected with control rAAV constructs. This finding might pinpoint to a vital and yet unknown contribution of HCN2 channels in establishing or maintaining the proper function of CA1 pyramidal neurons of the dorsal hippocampus.

Origins of eukaryotic excitability.

All living cells interact dynamically with a constantly changing world. Eukaryotes, in particular, evolved radically new ways to sense and react to their environment. These advances enabled new and more complex forms of cellular behaviour in eukaryotes, including directional movement, active feeding, mating, and responses to predation. But what are the key events and innovations during eukaryogenesis that made all of this possible? Here we describe the ancestral repertoire of eukaryotic excitability and discuss five major cellular innovations that enabled its evolutionary origin. The innovations include a vastly expanded repertoire of ion channels, the emergence of cilia and pseudopodia, endomembranes as intracellular capacitors, a flexible plasma membrane and the relocation of chemiosmotic ATP synthesis to mitochondria, which liberated the plasma membrane for more complex electrical signalling involved in sensing and reacting. We conjecture that together with an increase in cell size, these new forms of excitability greatly amplified the degrees of freedom associated with cellular responses, allowing eukaryotes to vastly outperform prokaryotes in terms of both speed and accuracy. This comprehensive new perspective on the evolution of excitability enriches our view of eukaryogenesis and emphasizes behaviour and sensing as major contributors to the success of eukaryotes.This article is part of the theme issue ‘Basal cognition: conceptual tools and the view from the single cell’.

Examining the Use, Perception, and Motivation of Cord-Cutting: A Consumer Segment Approach

This study investigates the factors affecting the cord-cutting behavior of media consumers. Adopting the frameworks of diffusion of innovations, uses and gratifications theory (U&G), channel repertoire, and media usage/segmentation, we conducted an online survey of 860 respondents in the United States to examine the use, perception, and motivation of cord-cutting behavior among three different consumer segments: cord-cutters, cord-loyalists, and cord-couplers. The results show that cord-cutting is predicted by eudaimonic and exploration motivators, perceived compatibility and complementarity, and age.

Optimized Tuning of Auditory Inner Hair Cells to Encode Complex Sound through Synergistic Activity of Six Independent K+ Current Entities

Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K+ conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K+ conductances mediated by Kv1.8, Kv7.4, Kv11.1, Kv12.1, and BKCa channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials.

MicroRNAs as regulators of brain function and targets for treatment of epilepsy.

Seizures result from hypersynchronous, abnormal firing of neuronal populations and are the primary clinical symptom of the epilepsies. Brain tissue from animal models and patients with acquired forms of epilepsy commonly features selective neuronal loss, gliosis, inflammatory markers and microscopic and macroscopic reorganization of networks. The gene expression landscape is a critical driver of these changes, and gene expression is fine tuned by small, non-coding RNAs called microRNAs (miRNAs). miRNAs inhibit the function of protein-coding transcripts, resulting in changes in multiple aspects of cell structure and function, including axonal and dendritic structure and the repertoire of neurotransmitter receptors, ion channels and transporters that establish neurophysiological functions. Dysregulation of the miRNA system has emerged as a mechanism that underlies epileptogenesis. Given that miRNAs can act on multiple mRNA targets, their manipulation offers a novel, multi-targeting approach to correct disturbed gene expression patterns. Targeting of some miRNAs has also been used to selectively upregulate individual transcripts, offering the possibility of precision therapy approaches for disorders of haploinsufficiency. In this Review, we discuss how miRNAs determine and control neuronal and glial functions, how this process is altered in states associated with hyperexcitability, and the prospects for miRNA targeting for the treatment of epilepsy.

Pharmacological Profile of the Sodium Current in Human Stem Cell-Derived Cardiomyocytes Compares to Heterologous Nav1.5+β1 Model.

The cardiac Nav1.5 mediated sodium current (INa) generates the upstroke of the action potential in atrial and ventricular myocytes. Drugs that modulate this current can therefore be antiarrhythmic or proarrhythmic, which requires preclinical evaluation of their potential drug-induced inhibition or modulation of Nav1.5. Since Nav1.5 assembles with, and is modulated by, the auxiliary β1-subunit, this subunit can also affect the channel’s pharmacological response. To investigate this, the effect of known Nav1.5 inhibitors was compared between COS-7 cells expressing Nav1.5 or Nav1.5+β1 using whole-cell voltage clamp experiments. For the open state class Ia blockers ajmaline and quinidine, and class Ic drug flecainide, the affinity did not differ between both models. For class Ib drugs phenytoin and lidocaine, which are inactivated state blockers, the affinity decreased more than a twofold when β1 was present. Thus, β1 did not influence the affinity for the class Ia and Ic compounds but it did so for the class Ib drugs. Human stem cell-derived cardiomyocytes (hSC-CMs) are a promising translational cell source for in vitro models that express a representative repertoire of channels and auxiliary proteins, including β1. Therefore, we subsequently evaluated the same drugs for their response on the INa in hSC-CMs. Consequently, it was expected and confirmed that the drug response of INa in hSC-CMs compares best to INa expressed by Nav1.5+β1.

Single-cell transcriptomes and whole-brain projections of serotonin neurons in the mouse dorsal and median raphe nuclei.

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.