Parallel neural pathways control sodium consumption and taste valence.

The hedonic value of salt fundamentally changes depending on the internal state. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive , whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.

Open-state structure and pore gating mechanism of the cardiac sodium channel.

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.

Neural Control and Modulation of Thirst, Sodium Appetite, and Hunger.

The function of central appetite neurons is instructing animals to ingest specific nutrient factors that the body needs. Emerging evidence suggests that individual appetite circuits for major nutrients-water, sodium, and food-operate on unique driving and quenching mechanisms. This review focuses on two aspects of appetite regulation. First, we describe the temporal relationship between appetite neuron activity and consumption behaviors. Second, we summarize ingestion-related satiation signals that differentially quench individual appetite circuits. We further discuss how distinct appetite and satiation systems for each factor may contribute to nutrient homeostasis from the functional and evolutional perspectives.

Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

Voltage Sensing Comes to Rest.

Voltage sensing by ion channels is the key event enabling the generation and propagation of electrical activity in excitable cells. In this issue of Cell, Wisedchaisri et al. provide a structural view of a voltage-gated sodium channel in its resting closed conformation.

Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel.

Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.

Structure of an Ancient Respiratory System.

Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing complex I are homologous respiratory complexes with a common ancestor, but a structural basis for their evolutionary relationship is lacking. Here, we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophile Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of complex I while its membrane-embedded ion-translocation module can be divided into a H+- and a Na+-translocating unit. The H+-translocating unit is rotated 180° in-membrane with respect to its counterpart in complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na+-translocating unit, absent in complex I, resembles that found in the Mrp H+/Na+ antiporter and enables hydrogen gas evolution by MBH to establish a Na+ gradient for ATP synthesis near 100°C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.

Cryo-EM Captures the Dynamics of Ion Channel Opening.

In this issue of Cell, Hite and MacKinnon (2017) report the open conformation structure of Slo2.2, a neuronal Na+-activated K+ channel. More importantly, 3D classification of electron cryomicroscopy (cryo-EM) images allows visualization of the structural transition that occurs as the open probability of individual channels increases with added sodium.

Structural Titration of Slo2.2, a Na+-Dependent K+ Channel.

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.

Sex differences in blood pressure regulation and hypertension: renal, hemodynamic, and hormonal mechanisms.

The teleology of sex differences has been argued since at least as early as Aristotle's controversial Generation of Animals more than 300 years BC, which reflects the sex bias of the time to contemporary readers. Although the question "why are the sexes different" remains a topic of debate in the present day in metaphysics, the recent emphasis on sex comparison in research studies has led to the question "how are the sexes different" being addressed in health science through numerous observational studies in both health and disease susceptibility, including blood pressure regulation and hypertension. These efforts have resulted in better understanding of differences in males and females at the molecular level that partially explain their differences in vascular function and renal sodium handling and hence blood pressure and the consequential cardiovascular and kidney disease risks in hypertension. This review focuses on clinical studies comparing differences between men and women in blood pressure over the life span and response to dietary sodium and highlights experimental models investigating sexual dimorphism in the renin-angiotensin-aldosterone, vascular, sympathetic nervous, and immune systems, endothelin, the major renal sodium transporters/exchangers/channels, and the impact of sex hormones on these systems in blood pressure homeostasis. Understanding the mechanisms governing sex differences in blood pressure regulation could guide novel therapeutic approaches in a sex-specific manner to lower cardiovascular risks in hypertension and advance personalized medicine.

Navigating the multifaceted intricacies of the Na+-Cl- cotransporter, a highly regulated key effector in the control of hydromineral homeostasis.

The Na+-Cl- cotransporter (NCC; SLC12A3) is a highly regulated integral membrane protein that is known to exist as three splice variants in primates. Its primary role in the kidney is to mediate the cosymport of Na+ and Cl- across the apical membrane of the distal convoluted tubule. Through this role and the involvement of other ion transport systems, NCC allows the systemic circulation to reclaim a fraction of the ultrafiltered Na+, K+, Cl-, and Mg+ loads in exchange for Ca2+ and [Formula: see text]. The physiological relevance of the Na+-Cl- cotransport mechanism in humans is illustrated by several abnormalities that result from NCC inactivation through the administration of thiazides or in the setting of hereditary disorders. The purpose of the present review is to discuss the molecular mechanisms and overall roles of Na+-Cl- cotransport as the main topics of interest. On reading the narrative proposed, one will realize that the knowledge gained in regard to these themes will continue to progress unrelentingly no matter how refined it has now become.

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.

A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability.

Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

The cellular mechanisms of neuronal swelling underlying cytotoxic edema.

Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema.

Structure of the human dopamine transporter in complex with cocaine.

The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine1. As part of the neurotransmitter sodium symporter family, DAT uses the Na+ gradient across cell membranes to transport dopamine against its chemical gradient2. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na+ occupies the second Na+ site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na+ space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.

Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Molecular basis of human noradrenaline transporter reuptake and inhibition.

Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types1. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane2. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.

Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.

T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.

Mechanosensitive Ion Channels: Structural Features Relevant to Mechanotransduction Mechanisms.

Activation of mechanosensitive ion channels underlies a variety of fundamental physiological processes that require sensation of mechanical force. Different mechanosensitive channels adapt distinctive structures and mechanotransduction mechanisms to fit their biological roles. How mechanosensitive channels work, especially in animals, has been extensively studied in the past decade. Here we review key findings in the functional and structural characterizations of these channels and highlight the structural features relevant to the mechanotransduction mechanism of each specific channel.