Conductance selectivity of Na+ across the K+ channel via Na+ trapped in a tortuous trajectory

Ion selectivity of the potassium channel is crucial for regulating electrical activity in living cells; however, the mechanism underlying the potassium channel selectivity that favors large K⁺ over small Na⁺ remains unclear. Generally, Na⁺ is not completely excluded from permeation through potassium channels. Herein, the distinct nature of Na⁺ conduction through the prototypical KcsA potassium channel was examined. Single-channel current recordings revealed that, at a high Na⁺ concentration (200 mM), the channel was blocked by Na⁺, and this blocking was relieved at high membrane potentials, suggesting the passage of Na⁺ across the channel. At a 2,000 mM Na⁺ concentration, single-channel Na⁺ conductance was measured as one-eightieth of the K⁺ conductance, indicating that the selectivity filter allows substantial conduit of Na⁺. Molecular dynamics simulations revealed unprecedented atomic trajectories of Na⁺ permeation. In the selectivity filter having a series of carbonyl oxygen rings, a smaller Na⁺ was distributed off-center in eight carbonyl oxygen-coordinated sites as well as on-center in four carbonyl oxygen-coordinated sites. This amphipathic nature of Na⁺ coordination yielded a continuous but tortuous path along the filter. Trapping of Na⁺ in many deep free energy wells in the filter caused slow elution. Conversely, K⁺ is conducted via a straight path, and as the number of occupied K⁺ ions increased to three, the concerted conduction was accelerated dramatically, generating the conductance selectivity ratio of up to 80. The selectivity filter allows accommodation of different ion species, but the ion coordination and interactions between ions render contrast conduction rates, constituting the potassium channel conductance selectivity.

Ion selectivity is a fundamental property of ion channels that generate the physiological functions of the cell membrane. Ion selectivity determines the direction of net currents through a channel under a given ionic composition of intracellular and extracellular solutions (1). Potassium channels carry outward K⁺ currents even in the presence of abundant Na⁺ ions in the extracellular solution. The selective passage of large K⁺ ions (ionic radius of 1.3 Å) over smaller Na⁺ ions (1.0 Å) (2–5) is a feature of the potassium channel that distinguishes it from other channels. The molecular mechanisms underlying this selectivity have been studied extensively for decades (2, 4, 6–8) but remain to be fully elucidated (6, 9–11). To examine the selectivity mechanism, the KcsA potassium channel has been applied as a prototypical channel; a broad spectrum of data related to ion permeation and selectivity has been accumulated (8, 12–17). The crystal structure of the KcsA channel revealed that its selectivity filter is narrow (3 Å in diameter) and short (12 Å in length), which is common for all potassium channels.Generally, potassium channels share a typical selectivity feature for monovalent cations, and the permeability ratio relative to K⁺, which is the most frequently used parameter for the ion selectivity, is plotted here as a function of the ionic radius of the relevant ion species (Fig. 1) (1, 4). The channel allows the permeation of ionic species in a limited window with respect to ion size. Ionic species, with ionic radii ranging from 0.9 to 1.7 Å, including K⁺, Tl⁺, Rb⁺, and Na⁺ as a limiting case, are permissible for conduction; however, larger and even a smaller ion (Li⁺) are rejected from permeation. This feature of the potassium channel has been explained by the classical and static concept of the snug fit (3, 18), wherein an ion species within a limited ion size is selected through matching to the cavity size in the pore via formation of a host-guest complex (19). However, molecular dynamics (MD) simulation has demonstrated that the filter structure of the potassium channel is intrinsically flexible, dismissing the strict cavity size (14). Moreover, the crystal structure revealed that even the nonconducting Li⁺ is bound in the selectivity filter (12). A more dynamic picture of the cavity, such as that based on the concept of strain energy, was proposed (10, 14, 19).Open in a separate windowFig. 1.Ion selectivity of potassium channels. Permeability ratios of monovalent cations relative to K⁺ as a function of ionic radius are shown for various types of potassium channels. The data were collected from literature (SI Appendix, Table S1) and are presented as a box plot, where the box covers the 25th to 75th percentiles, with the center line indicating the median. The potassium channel selectivity is characterized as a band-pass filter with a limited ion-size window (broken green line). The band-pass is arbitrarily decomposed into plausible small-pass (or low-pass; broken blue line) and large-pass (or high-pass; broken red line) filters. The lines do not have physical meanings.In the selectivity filter, K⁺ is coordinated to eight carbonyl oxygens (cage configuration), whereas Na⁺ is coordinated to four carbonyl oxygens (plane configuration) (9, 12, 20–24). High-affinity K⁺ binding to the filter deduced from the equilibrium crystal structure and spectroscopy has been interpreted as the basis for strict K⁺ selectivity (25–28). However, a comparison between the affinity and conductance revealed that the high affinity of an ion is not a determinant of its selectivity (11, 29). Recently, data supporting the low-affinity binding of K⁺ to the filter have been accumulated (30–32), and a dynamic principle has been proposed as an alternative mechanism for the selectivity (20, 33). Simulation studies revealed differential conduction of K⁺ and Na⁺ under different energy profiles, and Burykin et al. demonstrated that a different effective charge–charge dielectric for the Na⁺ and K⁺ was a unifying idea of the origin of the selectivity (34–36). The distinct nature of Na⁺ from that of K⁺ under the collective dynamics of ions and water within the selectivity filter has been studied extensively (9, 12, 20, 21, 33, 37–40) (see references in the following review articles: refs. 6, 9–11, 33, 37).Here, we consider that the selectivity arises dynamically and that traveling ions undergo multiple selection steps along the passage of the entire pore. To delineate these selectivity processes, we describe the unique selectivity of potassium channels as a feature of a band-pass filter. The band-pass filter is an electrical device, allowing the passage of signals within a limited frequency range by rejecting low- and high-frequency signals (41). By analogy, potassium channel selectivity is featured as a band-pass filter with respect to the ion size (broken green line, Fig. 1). Generally, a band-pass filter is fabricated from a combination of low- and high-pass filters. Accordingly, the band-pass feature of ion selectivity can be deconvoluted into two types of successive filtering processes along the pore. As small-pass (low-pass) filters (broken blue line, Fig. 1), potassium channels allow the passage of small ionic species with a size cutoff of ∼1.7 Å. The working principle of the small-pass filter is shared with that of other types of channels and is determined by the geometrical pore size (1, 42). Simultaneously, potassium channels impose a unique large-pass (high-pass) filter (broken red line, Fig. 1) that rejects small ions; this is the main issue addressed and studied herein.Na⁺ is a small ion that can serve as a signature ion to characterize the features of conduction through potassium channels. Consequently, in this study, Na⁺ conduction was examined using single-channel current recordings and MD simulations for the KcsA potassium channel. To characterize the selectivity, the permeability ratio obtained from experimentally measured reversal potentials has frequently been used, as in Fig. 1. Although it is an experimentally feasible parameter, the underlying Nernst–Planck equation assumes independent ion diffusion across a homogeneous membrane phase rather than through a structured pore (1, 43). The reversal potential for calculating the permeability ratio is simply the membrane potential at which inward currents and outward currents are balanced (zero-current potential). Alternatively, the single-channel conductance ratio at a specific membrane potential for different ion species is more straightforward and contrasts the integrated permeation kinetics of different ion species through the pore (4). However, the single-channel conductance of Na⁺ through the potassium channel has not been measured (12, 20, 33) due to experimental difficulty. Instead, indirect evidence of Na⁺ conduction through a potassium channel at the single-channel level has been reported as a punchthrough (12, 13). In the previous simulation, Na⁺ and K⁺ conduction through the KcsA channel was performed, and the conductance ratio was theoretically predicted (34).In the present study, the intracellular Na⁺ concentration was increased, exceeding the physiological concentration range, and the distinct Na⁺ processes in the pore, which involved blocking and a single-channel Na⁺ current through the potassium channel, were resolved. MD simulations revealed an intricate process of Na⁺ conductance at an atomic scale across the channel. Accordingly, critical permeation processes of the large-pass filter for Na⁺ conduction have been highlighted here.     

The amino-terminal domain of GluA1 mediates LTP maintenance via interaction with neuroplastin-65

Long-term potentiation (LTP) has long been considered as an important cellular mechanism for learning and memory. LTP expression involves NMDA receptor-dependent synaptic insertion of AMPA receptors (AMPARs). However, how AMPARs are recruited and anchored at the postsynaptic membrane during LTP remains largely unknown. In this study, using CRISPR/Cas9 to delete the endogenous AMPARs and replace them with the mutant forms in single neurons, we have found that the amino-terminal domain (ATD) of GluA1 is required for LTP maintenance. Moreover, we show that GluA1 ATD directly interacts with the cell adhesion molecule neuroplastin-65 (Np65). Neurons lacking Np65 exhibit severely impaired LTP maintenance, and Np65 deletion prevents GluA1 from rescuing LTP in AMPARs-deleted neurons. Thus, our study reveals an essential role for GluA1/Np65 binding in anchoring AMPARs at the postsynaptic membrane during LTP.

In 1973, Bliss and Lomo published the first observation of long-term potentiation (LTP) in which a tetanic stimulus caused a prolonged enhancement of synaptic transmission in rabbit hippocampus (1). Numerous studies have since demonstrated that LTP contributes to the neuronal mechanisms underlying learning and memory (2–4). The classic NMDA receptor (NMDAR)-dependent LTP is found in many brain regions and is studied mostly in hippocampal CA1 synapses (5, 6). Mechanistically, LTP can be divided into two sequential phases: initiation and maintenance. During LTP initiation, tetanic stimulation activates NMDARs that mediate rapid Ca²⁺ influx into dendritic spines, resulting in CaMKII activation, which subsequently recruits more AMPA-type glutamate receptors (AMPARs) into synapses, thus strengthening AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) (7, 8). LTP maintenance is thought to require the newly recruited AMPARs to remain at the postsynaptic membrane for an extended period of time, a process called synaptic trapping (9).AMPAR cellular trafficking, synapse anchoring, and synaptic function are dependent on the subunit composition of the core functional ion channel, which consists of a tetramer of subunits GluA1-GluA4. Each subunit consists of an amino-terminal domain (ATD, also known as N-terminal domain), a ligand-binding domain, four membrane-spanning segments, and an intracellular C-terminal domain (CTD). In mouse hippocampal CA1 pyramidal neurons, the most common types of AMPAR subunits are GluA1, GluA2, and GluA3 (10). Early studies using virus-based overexpression of the green fluorecent protein (GFP)-tagged AMPAR subunits suggest that GluA1 and GluA2 have differential trafficking capabilities in hippocampal neurons (11, 12); GluA2/A3 heteromers are constitutively trafficked to dendritic spines, while the synaptic cooperation of GluA1-containing AMPARs is dependent on neuronal activity. Thus, a subunit-specific model for AMPAR trafficking has been proposed (13), and LTP is thought to require certain sequences or domains of GluA1 (14). The emergent roles of the GluA1 CTD in synaptic plasticity have been extensively documented (13, 15–17). However, the observation that the CTD-lacking GluA1 is still present at the postsynaptic membrane and mediates LTP (18, 19) challenged the absolute requirement for GluA1 CTD in synaptic transmission and plasticity, indicating that other domains, such as the ATD, might have a previously uncovered role in LTP.Indeed, recent studies have revealed that the GluA1 ATD is required for synaptic transmission and LTP (20, 21). The ATD, which accounts for nearly half of the AMPAR coding sequence, projects nearly midway into the synaptic cleft where it may dynamically interact with proteins; such interactions might contribute to synaptic plasticity (22, 23). N-cadherin (24), a cell adhesion molecule, and neuronal pentraxins (25), secretory proteins, have been reported to associate with the ATDs of GluA2 and GluA4, respectively. However, whether GluA1 ATD has binding partners in the synaptic cleft remains unclear.In this study, we aimed to further understand the role of the GluA1 ATD in synaptic transmission and LTP and to investigate the molecular mechanism underlying its function. We found that the ATD is required for GluA1 synaptic function, both under basal conditions and during LTP. Furthermore, we have identified that GluA1 ATD directly interacts with neuroplastin-65 (referred to throughout as Np65), a single-transmembrane protein belonging to the immunoglobulin superfamily of cell adhesion molecules. Interaction of Np65 with the ATD of GluA1 is required for prolonged enhancement in synaptic transmission during LTP. Interestingly, it has been reported that as early as 20 y ago, Np65 antibody treatment causes impairment in LTP maintenance in hippocampal slices (26). Therefore, our results provide a molecular mechanism for GluA1- and Np65-mediated LTP maintenance.     

Selectivity filter ion binding affinity determines inactivation in a potassium channel

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Ion permeation gating within the selectivity filter (SF) of potassium (K⁺) channels has been proposed to control channel activity in different ways for different family members. In voltage-dependent K⁺ (K_V) channels, the SF has been proposed to underlie C-type inactivation (1–3), resulting in the progressive loss of current following the activation of a channel gate located near the intracellular side of the pore (4). C-type inactivation in K_V channels has been shown to be strongly dependent on the affinity of a particular binding site for permeant ions in the pore, and the affinity of these pore sites has been proposed to depend not only on the SF chemical composition, but also on regions outside of the SF (5–8). A structure-function model for this mechanism has been provided most specifically by studies of the proton-gated KcsA channel (9–11) where opening of the activation gate is correlated with a conformational constriction and a decrease in ion occupancy within the four K⁺ binding sites of the SF (named S1 to S4) (9, 12–15). Experimental and structural studies of KcsA in low K⁺ showed that the SF constriction consists of an outward flip of the carbonyl groups of the Gly77, in the middle of the signature sequence (TVGYG) of K⁺ channels, associated with a loss of K⁺ binding at site S2 of the SF (9, 14, 16–20). These conformational changes were accompanied by the binding of several water molecules behind the SF, stabilizing this constricted (also called flipped) state by sterically preventing the SF from switching back into its conductive state (16, 18–20). While some reports challenge this view (21, 22), this activation gate-coupled collapse of the SF is now generally accepted as the mechanism underlying C-type inactivation in K⁺ channels.Several types of ligand-dependent K⁺ channels, including those opened by binding Ca²⁺, such as the BK and MthK channels (23–27), do not exhibit traditional C-type inactivation, despite possessing an identical SF with K_V and KcsA channels. Furthermore, these channels have been proposed to actually gate at the SF (28–32) although a recent cryogenic electron microscopy (cryo-EM) structure of MthK in the absence of calcium (33) revealed a steric closure at the bundle crossing inner gate region, suggesting that there may be two gates involved in calcium gating. Nevertheless, at this time, the structural correlates of SF gating and the difference from inactivation are unknown.In the present study, we set out to first investigate whether we can capture different gating states of MthK by obtaining X-ray structures of its pore (Fig. 1A) in wide-ranging concentrations of K⁺. MthK channels have been previously shown to display a decrease in activity with depolarization, which is further augmented when external K⁺ concentration is lowered, a signature of SF gating and a hallmark of C-type inactivation (34). We reasoned that K⁺ titration of MthK pore structures may provide insights into the molecular causes for K⁺-dependent SF gating and will indicate whether it shares features with the C-type inactivation observed in KcsA (such as a collapsed SF). Unlike KcsA, MthK SF did not collapse in similarly low K⁺ concentrations, suggesting that the clue to why MthK does not display traditional C-type inactivation may lie in understanding the molecular underpinnings that contribute to SF conformational change. Thus, we next investigated the dependence of SF conformation on ion occupancy and used molecular dynamics (MD) simulations to reveal a uniquely low affinity central S2 site in MthK, which may play a lead role in the SF-based channel closure. Overall, our results illustrate how the exact same sequence and structure of the SF in a K⁺ channel can lead to slight variations in K⁺ binding site chemistry, which in turn can lead to distinct functional phenotypes.Open in a separate windowFig. 1.Structure of the MthK pore in different K⁺ concentrations. (A) Overall architecture of wild-type MthK pore structure (three subunits of the tetrameric pore shown for clarity) crystallized with 150 mM K⁺. The SF is highlighted by dashed lines. Alignment of this structure with that crystallized in 6 mM K⁺ yields an all-atom root-mean-square deviation (RMSD) value of 0.25 Å. (B) K⁺-omit electron density maps (2F_o – F_c contoured at 2.0 σ) for SF atoms from two opposing subunits. Structures were solved in 150, 50, 11, and 6 mM [K⁺], as indicated. Crystallographic statistics are in SI Appendix, Table S1. (C) MD system with the MthK (ribbons) embedded in a lipid bilayer (gray sticks) bathed in 200 mM KCl (K⁺ as green spheres, Cl⁻ as blue spheres, and water as red and white sticks).     

Ultrasound activates mechanosensitive TRAAK K+ channels through the lipid membrane

Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K⁺ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.

Manipulating cellular electrical activity is central to basic research and is clinically important for the treatment of neurological disorders including Parkinson’s disease, depression, epilepsy, and schizophrenia (1–4). Optogenetics, chemogenetics, deep brain stimulation (DBS), transcranial electrical stimulation, and transcranial magnetic stimulation are widely utilized neuromodulatory techniques, but each is associated with physical or biological limitations (5). Transcranial stimulation affords poor spatial resolution; deep brain stimulation and optogenetic manipulation typically require surgical implantation of stimulus delivery systems, and optogenetic and chemogenetic approaches necessitate genetic targeting of light- or small-molecule–responsive proteins.Ultrasound was first recognized to modulate cellular electrical activity almost a century ago, and ultrasonic neuromodulation has since been widely reported in the brain, peripheral nervous system, and heart of humans and model organisms (5–12). Ultrasonic neuromodulation has garnered increased attention for its advantageous physical properties. Ultrasound penetrates deeply through biological tissues and can be focused to sub-mm (3) volumes without transferring substantial energy to overlaying tissue, so it can be delivered noninvasively, for example, to deep structures in the brain through the skull. Notably, ultrasound generates excitatory and/or inhibitory effects depending on the system under study and stimulus paradigm (5, 13, 14).The mechanisms underlying the effects of ultrasound on excitable cells remain largely unknown (5, 13). Ultrasound can generate a combination of thermal and mechanical effects on targeted tissue (15, 16) in addition to potential off-target effects through the auditory system (17, 18). Thermal and cavitation effects, while productively harnessed to ablate tissue or transiently open the blood–brain barrier (19), require stimulation of higher power, frequency, and/or duration than typically utilized for neuromodulation (5). Intramembrane cavitation or compressive and expansive effects on lipid bilayers could generate nonselective currents that alter cellular electrical activity (5, 13). Alternatively, ultrasound could activate mechanosensitive ion channels through the deposition of acoustic radiation force that increases membrane tension or geometrically deforms the lipid bilayer (5, 15). Consistent with this notion, behavioral responses to ultrasound in Caenorhabditis elegans require mechanosensitive, but not thermosensitive, ion channels (20), and a number of mechanosensitive (and force-sensitive, but noncanonically mechanosensitive) ion channels have been implicated in cellular responses to ultrasound including two-pore domain K⁺ channels (K2Ps), Piezo1, MEC-4, TRPA1, MscL, and voltage-gated Na⁺ and Ca²⁺ channels (20–24, 25). Precisely how ultrasound impacts the activity of these channels is not known.To better understand mechanisms underlying ultrasonic neuromodulation, we investigated the effects of ultrasound on the mechanosensitive ion channel TRAAK (26, 27). K2P channels including TRAAK are responsible for so called “leak-type” currents because they approximate voltage- and time-independent K⁺-selective holes in the membrane, although more complex gating and regulation of K2P channels is increasingly appreciated (28, 29). TRAAK has a very low open probability in the absence of membrane tension and is robustly activated by force through the lipid bilayer (30–32). Mechanical activation of TRAAK involves conformational changes that prevent lipids from entering the channel to block K⁺ conduction (31). Gating conformational changes are associated with shape changes that expand the channel and make it more cylindrical in the membrane plane upon opening. These shape changes are energetically favored in the presence of membrane tension, resulting in a tension-dependent energy difference between states that favors channel opening (31). TRAAK is expressed in neurons and has been localized exclusively to nodes of Ranvier, the excitable action potential propagating regions of myelinated axons (33, 34). TRAAK is found in most (∼80%) myelinated nerve fibers in both the central and peripheral nervous systems, where it accounts for ∼25% of basal nodal K⁺ currents. As in heterologous systems, mechanical stimulation robustly activates nodal TRAAK. TRAAK is functionally important for setting the resting potential and maintaining voltage-gated Na⁺ channel availability for spiking in nodes; loss of TRAAK function impairs high-speed and high-frequency nerve conduction (33, 34). Changes in TRAAK activity therefore appear well poised to widely impact neuronal excitability.We find that low-intensity and short-duration ultrasound rapidly and robustly activates TRAAK channels. Activation is observed in patches from TRAAK-expressing Xenopus oocytes, in patches containing purified channels reconstituted into lipid membranes, and in TRAAK-expressing mouse cortical neurons. Single-channel recordings reveal that canonical mechanical and ultrasonic activation are accomplished through a shared mechanism. We conclude that ultrasound activates TRAAK through the lipid membrane, likely by increasing membrane tension to promote channel opening. This work demonstrates direct mechanical activation of an ion channel by ultrasound using purified and reconstituted components, is consistent with endogenous mechanosensitive channel activity underlying physiological effects of ultrasound, and provides a framework for the development of exogenously expressed sonogenetic tools for ultrasonic control of neural activity.     

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca²⁺-and voltage-activated K⁺ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2⁻ cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

The colonic epithelium is composed of a single layer of heterogeneous cells, covered by mucus, that separate the luminal contents from host tissues. Acting both in concert and individually, the diverse cells comprising the epithelial layer play the functions of protection (1), sensation (2, 3), transport of substances (4, 5), and repair (6). Colonic epithelial cells belong to three lineages: Absorptive enterocytes, enteroendocrine cells, and goblet cells (GCs). The colonic epithelium is morphologically organized into repeating units called crypts of Lieberkühn, where stem cells located at the base of the crypts divide and successively differentiate into the mature lineages as they migrate toward the crypt surface (7). Many of the key specialized functions of epithelial cells are, in part, defined by proteins involved in ion transport, located either on their luminal or basolateral membrane. Thus, among different gastrointestinal epithelial cells, ion channels, carriers, exchangers, and pumps work in concert to define a variety of essential functions: 1) Solute and electrolyte absorption and secretion in absorptive enterocytes (reviewed in refs. 5 and 8); 2) environment sensation and serotonin secretion by enteroendocrine cells (2, 9); and 3) mucus secretion by GCs and subsequent mucus maturation into the protective layer covering the epithelial surface (10–12). Despite this progress, ionic transport in GCs and its implications in GC physiology is a topic that remains poorly understood. Here, we address the role of the Ca²⁺- and voltage-activated K⁺ channel (BK channel) in GCs.GCs play two primary roles: One related to the maintenance of the mucosal barrier (reviewed in refs. 1 and 13) and one related with the mucosal immune homeostasis (reviewed in refs. 14 and 15). The role of GCs in barrier maintenance consists in generation of the mucus layer lining the intestinal lumen. One way GCs carry out this role is by secreting MUC2, the gel-forming mucin that forms the scaffold of the mucus layer separating luminal pathogens and commensal microbiota from the epithelial surface (11, 12, 15, 16). This separation is critical, as has been demonstrated in both animal models and humans: Mouse models with deficient mucus layer generation develop spontaneous colitis (16, 17), whereas a more penetrable mucus layer has been observed in patients with ulcerative colitis (UC), a form of human inflammatory bowel disease (IBD) (18, 19). The constant replenishment of the mucus layer involves MUC2 exocytosis from GCs, and subsequent maturation (hydration and expansion) of the secreted MUC2 to form the gel-like mucus coating the epithelium (15). Both exocytosis and maturation of MUC2 are highly dependent on anion and K⁺ transport (10–12, 20). It has been proposed that mucin exocytosis in colon requires activities of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) (20, 21), and also anion and K⁺ channels whose identities are still unclear (20). It is also not clearly known whether specific ionic conductances are intrinsic to GCs or are located in the surrounding absorptive enterocytes. Although several types of K⁺ channels—including K_Ca3.1, Kv7.1, and BK channels—have been found in colonic epithelial cells (22–27), to what extent any of those K⁺ channels are specifically associated with GCs or critical to their function remains unclear. To date, most functional studies about colonic K⁺ channels have focused on their roles in electrolyte and fluid secretion/absorption of the whole colon, whereas the cellular events relating K⁺ channels to specific roles in GC function are still poorly understood.Among colonic epithelial K⁺ channels, the BK channel (also known as K_Ca1.1), the Ca²⁺- and voltage-activated K⁺ channel of high conductance, has been proposed to be the main component of colonic K⁺ secretion into the lumen (28–30). BK channels are homotetramers of the pore-forming BKα subunit, but can also contain tissue-specific regulatory subunits that critically define the functional properties of the channel (31). BK channels composed exclusively of the pore-forming BKα subunit are unlikely to be activated at the physiological conditions of epithelial cells and, as a consequence, the molecular properties of colonic BK channels that would allow them to contribute to colonic ion transport remain unclear. Recently, we established that the leucine-rich repeat-containing protein 26 (LRRC26), a BK regulatory γ-subunit, is specifically expressed in secretory epithelial cells, including GCs of the gastrointestinal tract (32). When LRRC26 is present in a BK channel complex, the resulting channel activates near normal resting physiological conditions, even in the absence of any elevation of intracellular Ca²⁺ (33).In the present study, we have specifically probed the role of BK channels in cells of the colonic epithelium and examined the impact of deletions of either the BKα subunit or LRRC26 on colonic function. Here, through recordings from identified GCs in intact colonic crypts, we show that LRRC26-associated BK channels contribute the major K⁺ current at low intracellular Ca²⁺ (∼250 nM) in mouse colonic GCs. Furthermore, the LRRC26-containing BK channels are activated near −40 mV, even in the absence of intracellular Ca²⁺. In contrast, in identified GCs from Lrrc26^−/− mice, BK current is present, but it is only activated at membrane potentials unlikely to ever occur physiologically. Surprisingly, all colonic epithelial MUC2⁻ cells sampled completely lack functional BK channels. To establish that the LRRC26-containing BK channels contribute to normal K⁺ fluxes in intact colon tissue, we show that the transepithelial current across distal colon at rest has a component dependent on LRRC26-associated BK channels, which is absent when either BKα or LRRC26 is genetically deleted. Moreover, the genetic ablation of either LRRC26 or BK channel results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). Overall, our results suggest that normal potassium flux through LRRC26-associated BK channels in GCs has a protective role against development of colitis.     

PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana

Plant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO1 (PZO1) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO1 is expressed in the columella and lateral root cap cells of the root tip, which are known to experience robust mechanical strain during root growth. Deleting PZO1 from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild-type plants. pzo1 mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of PZO1 in root mechanotransduction. Finally, a chimeric PZO1 channel that includes the C-terminal half of PZO1 containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO1 plays an important role in root mechanotransduction and establish PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

Plants extend roots within the soil to access water and nutrients as well as provide stability for the aerial parts of the plant. Underground barriers caused by drought and/or heterogeneous soil components can exert mechanical resistance that alters root extension and penetration (1–3). The root cap at the very tip of the primary root is a dynamic organ that contains different classes of stem cells that divide asymmetrically and is essential for growth through harder media and soils (4). Bending or poking root tips elicits a transient Ca²⁺ influx with short latency that is blocked by lanthanides, including Gd³⁺, a nonselective inhibitor of mechanically activated (MA) cation channels (5–7). However, the molecular identity of putative ion channels underlying this response is unknown. Only a few mechanosensitive ion channels have been described in plants (8). MSL8 plays a mechanosensory role in pollen (9), MSL10 is involved in cell swelling (8, 10), OSCA1 has mainly been characterized for its role in osmosensation (11), and OSCA1.3 regulates stomatal closure during immune signaling (12). It has been proposed that MCA1, expressed in the elongation zone but not the root cap, is a stretch-activated calcium permeable ion channel involved in soil penetration; however, evidence for its being a bona fide ion channel capable of detecting mechanical force is lacking (13–15). Recently, it has been shown that mechanosensitive Ca²⁺ channel activity is dependent on the developmental regulator DEK1; however, whether DEK1 is a pore-forming ion channel has not yet been addressed (16, 17). The genome of Arabidopsis thaliana encodes an ortholog of the mammalian mechanosensitive ion channels PIEZO1 and PIEZO2 (18). Given that PIEZOs play prominent roles in multiple aspects of animal mechanosensation and physiology (19–22), we investigated the role of A. thaliana PIEZO1 (PZO1) in plant mechanosensation. A recent study reported that PZO1 regulated virus translocation within the plant, but its specific role in mechanotransduction was not addressed (23). Here we use genetic tools, electrophysiological methods, and calcium imaging to investigate the role of PZO1 in root mechanosensation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Nonthermal and reversible control of neuronal signaling and behavior by midinfrared stimulation

Various neuromodulation approaches have been employed to alter neuronal spiking activity and thus regulate brain functions and alleviate neurological disorders. Infrared neural stimulation (INS) could be a potential approach for neuromodulation because it requires no tissue contact and possesses a high spatial resolution. However, the risk of overheating and an unclear mechanism hamper its application. Here we show that midinfrared stimulation (MIRS) with a specific wavelength exerts nonthermal, long-distance, and reversible modulatory effects on ion channel activity, neuronal signaling, and sensorimotor behavior. Patch-clamp recording from mouse neocortical pyramidal cells revealed that MIRS readily provides gain control over spiking activities, inhibiting spiking responses to weak inputs but enhancing those to strong inputs. MIRS also shortens action potential (AP) waveforms by accelerating its repolarization, through an increase in voltage-gated K⁺ (but not Na⁺) currents. Molecular dynamics simulations further revealed that MIRS-induced resonance vibration of –C=O bonds at the K⁺ channel ion selectivity filter contributes to the K⁺ current increase. Importantly, these effects are readily reversible and independent of temperature increase. At the behavioral level in larval zebrafish, MIRS modulates startle responses by sharply increasing the slope of the sensorimotor input–output curve. Therefore, MIRS represents a promising neuromodulation approach suitable for clinical application.

Many forms of neuromodulation have been used for the regulation of brain functions and the treatment of brain disorders. Some physical approaches, such as electrical, magnetic, and optical (electromagnetic; EM) stimulation could be employed to manipulate neural spiking activity and achieve neuromodulation. Among them, deep-brain electrical stimulation has become a gold standard treatment for advanced Parkinson’s disease; transcranial magnetic stimulation also generates electrical current in selected brain regions and has been used for mood regulation. In contrast, optical neural stimulation has not been used clinically, largely due to the risk of tissue damage by overheating and unclear mechanisms. Although optogenetic manipulation and stimulation avoid these problems and show cell specificity, the requirement of expression of exogenous genes hinders its use in humans (1).Optical infrared neural stimulation (INS) is emerging as an area of interest for neuromodulation and potential clinical application. INS utilizes brief light pulses to activate excitable cells or tissues in the illumination spot. Previous studies showed that INS could activate peripheral nerves (2), peripheral sensory systems (3, 4), and cardiac tissue (5). In the central nervous system (CNS), initial studies found that INS could evoke neural responses in rat thalamocortical slices in vitro (6) and regulate spiking activity in rodent somatosensory cortex (7) and nonhuman primate visual cortex in vivo (8). Because of its high spatial precision, focal INS has been recently applied to map brain connectomes (9). The underlying mechanism of INS, however, remains poorly understood. The predominant view is the transduction of EM energy to thermal heat (10, 11) will excite the cell, possibly due to heat-induced transmembrane capacitive charge (12), changes in ion channel activity (13), or cell damage (14). Since previous studies tended to choose infrared wavelengths with high water absorption for efficient heat generation, it remains unclear whether INS exerts nonthermal effects on ion channel and neuronal spiking activity.While most studies on infrared stimulation have been conducted at near-infrared wavelengths, whether midinfrared wavelengths can regulate neural function is unknown. Because the frequency of midinfrared light falls into the frequency range of chemical bond vibration (15–17), nonlinear resonances may occur within biomolecules (18–20), leading to dramatic changes in their conformation and function (21) and thus producing nonthermal effects on biological systems. Ion channel proteins distributed on cell membranes could be potential molecular targets for midinfrared light. Among them, voltage-gated Na⁺ and K⁺ channels play critical roles in regulating the initiation and propagation of the action potential (AP), an all-or-none digital signal of neurons (22, 23). They also control the voltage waveform of the AP and thus the size of the postsynaptic response, ensuring analog-mode communication between neurons (24–27). It is of interest to know whether midinfrared stimulation (MIRS) can cause conformational change in these channel proteins and consequently regulate neuronal signaling. Previous studies revealed a low absorption of light by water in the midinfrared region from 3.5 to 5.7 μm (28), which could be a potential wavelength range for neuromodulation. Therefore, in this study, we explored whether MIRS with a specific wavelength in this range could exert nonthermal modulatory effects on channel activity, neuronal signaling, and behavior.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

Amygdala-hippocampal innervation modulates stress-induced depressive-like behaviors through AMPA receptors

Chronic stress is one of the most critical factors in the onset of depressive disorders; hence, environmental factors such as psychosocial stress are commonly used to induce depressive-​like traits in animal models of depression. Ventral CA1 (vCA1) in hippocampus and basal lateral amygdala (BLA) are critical sites during chronic stress-induced alterations in depressive subjects; however, the underlying neural mechanisms remain unclear. Here we employed chronic unpredictable mild stress (CUMS) to model depression in mice and found that the activity of the posterior BLA to vCA1 (pBLA-vCA1) innervation was markedly reduced. Mice subjected to CUMS showed reduction in dendritic complexity, spine density, and synaptosomal AMPA receptors (AMPARs). Stimulation of pBLA-vCA1 innervation via chemogenetics or administration of cannabidiol (CBD) could reverse CUMS-induced synaptosomal AMPAR decrease and efficiently alleviate depressive-like behaviors in mice. These findings demonstrate a critical role for AMPARs and CBD modulation of pBLA-vCA1 innervation in CUMS-induced depressive-like behaviors.

Major depression or major depressive disorder (MDD) is one of the most common and disabling mental disorders worldwide and is characterized by episodes of depressed mood, decreased drive, and loss of interest and pleasure (1, 2). Although MDD is multifactorial and heterogeneous by nature, negative stimuli such as stress are strongly implicated in MDD (3–5). The CA1 area in hippocampus has been one of the most intensively studied brain regions in depression research. Patients with MDD show a marked reduction in left CA1 volume (6). Rodents subjected to stress also have impaired spike timing-dependent long-term depression (LTD), as well as decreased spine density and down-regulated synaptic transmission in ventral CA1 (vCA1) pyramidal neurons (7, 8).vCA1 shares robust reciprocal projections with the basal lateral amygdala (BLA), a pivotal site underlying stress-induced emotional disorders. Based on morphological and genotypic distinction, the BLA has been divided into two subregions, anterior BLA (aBLA) and posterior BLA (pBLA) (9), both of which can be activated by negative (10–12) and positive (13, 14) emotion-associated events. Under physiological conditions, pBLA has a stronger monosynaptic connection to vCA1 than aBLA (15). Furthermore, optogenetic activation of the pBLA-vCA1 circuit can reduce anxiety-like behaviors (16). However, the mechanisms through which the pBLA-vCA1 circuit impacts the pathogenesis of depression remain unclear.AMPA receptors (AMPARs) play a critical role in synaptic plasticity, and dysfunction in AMPARs or proteins that regulate AMPAR trafficking has been linked to various neurological and psychiatric disorders (17). Stress selectively decreases expression of GluA1, an AMPAR subunit, in vCA1 and impairs AMPAR-mediated synaptic excitation (8). Antidepressants fluoxetine and reboxetine can increase the expression of GluA1/GluA3 in hippocampus and GluA2/GluA4 in prefrontal cortex (PFC), respectively (18). The cannabinoid system also plays an important role in regulation of mood and depression. Synthetic cannabinoids that activate cannabinoid type-1 (CB₁) and cannabinoid type-2 (CB₂) receptors can alleviate depressive-like symptoms in animal models (19, 20). Recently, cannabidiol (CBD), a nonpsychotomimetic component of Cannabis sativa, has emerged as a promising compound, since it exerts large-spectrum therapeutic potential in human mental disorders such as psychosis, anxiety, and depression (21). It has been reported that chronic CBD treatment induces behavioral and synaptic changes in stressed mice through CB₁/CB₂ receptor activation (22), but its acute antidepressant properties and underlying mechanisms have not been completely investigated.In the present study, we employed a chronic unpredictable mild stress (CUMS) procedure to induce a model of depression in mice and found that CUMS exposure resulted in decreased activity in pBLA-vCA1 innervation, while chemogenetic activation of pBLA-vCA1 input could reverse CUMS-induced behavior deficits. CUMS led to reduction in dendritic complexity, spine density, and synaptosomal AMPARs. Moreover, inhibition of AMPAR activity via application of DNQX under subthreshold stress conditions could induce depressive-like behaviors. Acute CBD administration reversed CUMS-induced reduction in mature spine density and synaptosomal AMPAR level in vCA1, and, more importantly, alleviated depressive-like behaviors. In summary, our study sheds light on the mechanisms underlying depressive-like behavior and advances our understanding of the complex pathology of depression.     

Selective tumor antigen vaccine delivery to human CD169+ antigen-presenting cells using ganglioside-liposomes

Priming of CD8⁺ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1⁺ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14⁺ CD169⁺ monocytes and Axl⁺ CD169⁺ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169⁺ moDCs and Axl⁺ CD169⁺ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8⁺ T cells. Finally, Axl⁺ CD169⁺ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169⁺ DCs to drive antitumor T cell responses.

The major breakthrough of immune-checkpoint inhibitors, such as anti-CTLA4 and anti–PD-L1, in cancer therapy is still limited to a minority of patients who respond to this treatment (1). Patients with pancreatic cancer, for example, failed to respond to monotherapies of checkpoint inhibitors in multiple trials (2, 3). Factors such as poor tumor immunogenicity, tumor-immunosuppressive microenvironment, and the lack of an existing tumor-specific immune response are thought to contribute to patients’ lack of response to these immune-checkpoint inhibitors (2, 4, 5). Nevertheless, the abundance of intratumoral CD8⁺ T cells is associated with longer survival of pancreatic cancer patients, suggesting these patients may benefit from a better antitumor immunity (6–8). Therefore, new strategies aiming to boost patients’ antitumor CD8⁺ T cell responses should be explored to improve current therapies.Dendritic cells (DCs) play a crucial role in eliciting immune responses against tumor-specific antigens and have therefore generated significant interest as a therapeutic target in the context of cancer immunotherapy (9). The most commonly used DC-based immunotherapy utilizes monocyte-derived DCs (moDCs) due to the large numbers that can be generated ex vivo. In general, moDC-based vaccines have shown some survival benefit and appear to be well-tolerated; however, the objective response rate in most studies is still relatively low (9, 10). Moreover, since generating DCs ex vivo is a laborious, time-consuming, and costly process, research is shifting toward targeting tumor antigens to naturally circulating or tissue-resident DCs in vivo as a vaccine strategy to induce immune responses (11). Both in mice and humans, DCs can be divided into several subsets, of which the conventional DCs (CD141⁺ cDC1 and CD1c⁺ cDC2) have been shown to be responsible for T cell priming (12, 13).In vivo DC targeting can be achieved by using antibodies or ligands that bind to DC-specific receptors and are directly conjugated to tumor antigen or to nanoparticles harboring tumor antigen. Targeting C-type lectin receptors in particular, such as DEC-205, Clec-9A, and DC-SIGN, has been demonstrated to induce antigen-specific and antitumor responses in mouse and human models (14–17). Recently, we compared two vaccination strategies of antigen–antibody conjugates directed to either DEC-205⁺ DCs or to CD169⁺ macrophages, a type of macrophage that acts as sentinel in secondary lymphoid organs (18). Remarkably, we observed that antigen targeting toward CD169⁺ macrophages led to a significant antigen-specific CD8⁺ T cell response that was as efficient as DEC-205 targeting and capable of suppressing tumor cell outgrowth (18–20). Stimulation of antigen-specific immune responses by targeting to CD169 has also been demonstrated using HLA-A2.1 transgenic mice and human CD169-expressing moDCs (21), indicating the immunotherapy potential of antigen targeting to CD169.In a resting state, CD169/Siglec-1 is highly expressed by a specific subtype of macrophages that are located bordering the marginal zone in the spleen and the subcapsular sinus of lymph nodes (22, 23). Their strategic location allows them to be among the first cells to encounter and to capture blood and lymph-borne pathogens, and, in conjunction with DCs, to initiate the appropriate immune responses (18, 19, 24, 25). In addition to combating infection, CD169⁺ macrophages have been implicated in antitumor immunity. They have been shown to capture tumor-derived materials in mouse and human (26, 27), and their frequency in tumor-draining lymph nodes is clearly associated with better clinical outcomes in several types of cancer (28–30). Although the exact mechanism is unclear, these observations suggest that lymphoid-resident CD169⁺ macrophages can positively contribute to antitumor immunity. Next to lymphoid tissue-resident macrophages, CD169 is also constitutively expressed by a recently described Axl⁺ Siglec6⁺ DC subset (Axl⁺ DCs, AS DCs, or pre-DCs) present in peripheral blood and lymphoid tissues (31–34). Axl⁺ DCs have been proposed as a distinct DC subset that has the capacity to produce inflammatory cytokines and to stimulate CD4⁺ and CD8⁺ T cells (31–33). In addition to these constitutively CD169-expressing macrophages and DCs, during inflammatory conditions, monocytes can up-regulate CD169 in response to type I interferons (IFN-Is) (35, 36).CD169 is a member of the sialic acid-binding Ig-like lectin (Siglec) receptor family that recognizes sialic acids present on glycoproteins or glycolipids on the cell surface and mediates cell–cell interactions and adhesion (37). Sialic acid-containing glycosphingolipids, such as GM3, GT1b, and GD1a gangliosides, are known to be endogenous ligands for CD169 molecules (38, 39). However, the CD169–sialic acid axis can be hijacked as a receptor entry molecule by viral pathogens, including murine leukemia virus (MLV), HIV, and Ebola virus to infect DCs or macrophages (40–43). The CD169-mediated entry and transinfection is dependent on gangliosides, including GM3, that are present on the viral lipid membrane (40, 44, 45). Interestingly, Axl⁺ DCs have been recently demonstrated to be the predominant DC subset to capture HIV in a CD169-dependent manner.In this study, we aimed to exploit ganglioside–CD169 interactions to develop a novel tumor antigen vaccination strategy that directs tumor antigens to human CD169⁺ antigen-presenting cells (APCs) using liposomes containing gangliosides. We generated liposomes with different types of gangliosides and assessed the binding and uptake by different types of human CD169⁺ APCs, including monocytes and primary and monocyte-derived macrophages and DCs. High-dimensionality mapping revealed the specificity of ganglioside-liposome targeting exclusively to circulating CD169⁺ monocytes and Axl⁺ DCs. To determine the efficacy of ganglioside-liposomes for antigen presentation, we encapsulated peptides derived from the pancreatic cancer-associated tumor antigen Wilms tumor 1 (WT1) or melanoma-associated gp100 antigen into the ganglioside-liposomes. CD169⁺ moDCs and Axl⁺ DCs loaded with these ganglioside-liposomes efficiently activated CD8⁺ T cells specific for these epitopes. Moreover, Axl⁺ DCs were present in patients with four different cancers and could be targeted by ganglioside-liposomes. Our data demonstrate that ganglioside-liposomes can be used as nanovaccine carriers that efficiently target CD169⁺ DCs for cross-presentation and antigen-specific T cell activation. In conclusion, our studies support the concept that cancer vaccines targeting to CD169 can be applied to boost CD8⁺ T cell responses in cancer patients.     

From the Cover: A moisture-enabled fully printable power source inspired by electric eels

Great efforts have been made to build integrated devices to enable future wearable electronics; however, safe, disposable, and cost-effective power sources still remain a challenge. In this paper, an all-solid-state power source was developed by using graphene materials and can be printed directly on an insulating substrate such as paper. The design of the power source was inspired by electric eels to produce programmable voltage and current by converting the chemical potential energy of the ion gradient to electric energy in the presence of moisture. An ultrahigh voltage of 192 V with 175 cells in series printed on a strip of paper was realized under ambient conditions. For the planar cell, the mathematical fractal design concept was adapted as printed patterns, improving the output power density to 2.5 mW cm⁻³, comparable to that of lithium thin-film batteries. A foldable three-dimensional (3D) cell was also achieved by employing an origami strategy, demonstrating a versatile design to provide green electric energy. Unlike typical batteries, this power source printed on flexible paper substrate does not require liquid electrolytes, hazardous components, or complicated fabrication processes and is highly customizable to meet the demands of wearable electronics and Internet of Things applications.

Future electronics call for wearable and even disposable power sources that can be customized to meet the demands of integrated systems; for example, flexible and disposable power sources are needed to provide energy for multifunctional contact lenses in real-time health monitoring applications (1). Among numerous manufacturing methods, printing shows great potential to build freeform products due to its geometric controllability, process flexibility, and cost-effectiveness (2). Printed energy storage devices such as lithium-ion batteries (3, 4) and alkaline batteries (5, 6) have been extensively studied to power next-generation devices. However, most of these energy storage systems contain hazardous or flammable components and thus cannot meet the safety and environmental requirements for green power sources, which are crucial for future disposable electronic devices.Nature gives inspiration for many ideas in the search of alternative green power sources (7). Among numerous forms of energy in nature, the chemical potential energy of the ion gradient is the foundation of many living species and has attracted increasing research attention since the award of the 2003 Nobel Prize in Chemistry for ion channels in cell membranes (8). The electric eel is an excellent example of an electric power source utilizing ion gradients to generate high voltages and currents. As shown in Fig. 1A, each electrocyte in the electric organ has highly selective membranes that can produce transmembrane potential via the flux of small ions. The action potential from the Na⁺ and K⁺ ion gradient is 65 and 85 mV, respectively, which adds up in series and results in a total transcellular potential of ∼150 mV (9). Thousands of electrocytes in series and parallel arrangement can generate potential up to 600 V and peak current of 1 A (10). To extract electric energy from the ion gradient, various energy systems have been designed such as reverse electrodialysis (RED), which can extract electric energy from the mixing of sea and river water (11, 12). However, these RED systems usually rely on bulky pumping systems and large ion-exchange membrane stacks and thus are not suitable for portable applications. Recently, some novel power sources have been developed to utilize the chemical potential energy of the ion gradient, such as artificial electric organs (10), nanofluidic devices (13), and moist-electric generators (14, 15). Although some of these devices are portable, they still suffer from low voltage output or require a complicated fabrication process.Open in a separate windowFig. 1.Schematic of structures of the electric eel’s electrocytes and the moisture-enabled power source. (A) Each electrocyte of the electric eel can generate 150 mV when stimulated via ion transportation of K⁺ and Na⁺ through highly selective ion channels on cell membranes. (B) The designed power source is composed of GO inks and rGO inks with different ion concentrations and a pair of silver electrodes. In the presence of moisture, the chemical potential energy of the ion gradient is converted to electric energy via directional ion migration and redox reactions and thus produces electric power.Herein, inspired by electric eels, we developed a fully printable all-solid-state power source based on graphene inks with an open-circuit voltage (OCV) up to 1.2 V for each cell, which is eight times higher than that of biological electric signals (9). The high voltage comes from the large cation concentration difference which is maintained by graphene oxide (GO) materials. GO materials are reported to have large salt intake, leading to highly concentrated solutions that are close to the saturation inside graphene capillaries, enable ultrafast ion permeation (16), and have been applied in adsorption (17) and selectively permeable membranes (18, 19). GO materials also have unimpeded water permeation capability (20), tunable ion-exchange properties (16, 21), and enhanced cation conductivity (22, 23) due to their unique two-dimensional (2D) nanofluidic channels, which are favorable for solid-state ionic conductors in humid environments. In the presence of water molecules, cations are transported from the high-concentration side to the low-concentration side, creating a potential difference. This directional ion migration is then converted to electron transportation at the surface of electrodes via redox reactions or charge adsorption, thus generating electric current output (Fig. 1B). When stored in low-moisture conditions, such as in a vacuum bag, a sealed container with desiccant and filled with dry N₂, or a climate-controlled facility with low humidity levels, there is limited ion transportation and therefore little to no self-discharge, ensuring a long shelf life unlike typical batteries (24). The cell is safe, disposable, and cost-effective, since all parts are inkjet printed onto paper to form an all-solid-state flexible power source, avoiding flammable and hazardous components. The geometric controllability of inkjet printing also allows a flexible cell pattern design (2). By optimizing the printing parameters, an ultrahigh voltage of 192 V was achieved on a small strip of paper under ambient conditions by connecting 175 cells in series. By adapting the mathematical space-filling curves as the printed pattern to increase the cell length (25, 26), the planar cell can achieve a short-circuit current of 170 μA and the power density can reach 2.5 mW cm⁻³ with energy density up to 0.41 mWh cm⁻³, which is comparable to that of lithium thin-film batteries (27). The output voltage and current are readily adjustable by arranging well-defined patterns in parallel and series connections. A foldable three-dimensional (3D) cell using an origami pattern was also fabricated, demonstrating a versatile design strategy for printed electronics. This interdisciplinary work opens a novel design direction for utilizing ion gradients and offers a simple but effective method to provide scalable green electric power for practical applications.     

Neuromodulator release in neurons requires two functionally redundant calcium sensors

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

To date, over 100 genes encoding neuropeptides and neurotrophic factors, together referred to as neuromodulators, are identified, and most neurons express neuromodulators and neuromodulator receptors (1). Neuromodulators travel through neurons in dense core vesicles (DCVs) and, upon secretion, regulate neuronal excitability, synaptic plasticity, and neurite outgrowth (2–4). Dysregulation of DCV secretion is linked to many brain disorders (5–7). However, the molecular mechanisms that regulate neuromodulator secretion remain largely elusive.Neuromodulator secretion, like neurotransmitter secretion from synaptic vesicles (SVs), is tightly controlled by Ca²⁺. The Ca²⁺ sensors that regulate secretion have been described for other secretory pathways but not for DCV exocytosis in neurons. Synaptotagmin (Syt) and Doc2a/b are good candidate sensors due to their interaction with SNARE complexes, phospholipids, and Ca²⁺ (8–11). The Syt family consists of 17 paralogs (12, 13). Eight show Ca²⁺-dependent lipid binding: Syt1 to 3, Syt5 to 7, and Syt9 and 10 (14, 15). Syt1 mediates synchronous SV fusion (8), consistent with its low Ca²⁺-dependent lipid affinity (15, 16) and fast Ca²⁺/membrane dissociation kinetics (16, 17). Syt1 is also required for the fast fusion in chromaffin cells (18) and fast striatal dopamine release (19). Synaptotagmin-7 (Syt7), in contrast, drives asynchronous SV fusion (20), in line with its a higher Ca²⁺ affinity (15) and slower dissociation kinetics (16). Syt7 is also a major calcium sensor for neuroendocrine secretion (21) and secretion in pancreatic cells (22–24). Other sensors include Syt4, which negatively regulates brain-derived neurothropic factor (25) and oxytocin release (26), in line with its Ca²⁺ independency. Syt9 regulates hormone secretion in the anterior pituitary (27) and, together with Syt1, secretion from PC12 cells (28, 29). Syt10 controls growth factor secretion (30). However, Syt9 and Syt10 expression is highly restricted in the brain (31–33). Hence, the calcium sensors for neuronal DCV fusion remain largely elusive. Because DCVs are generally not located close to Ca²⁺ channels (34), we hypothesized that DCV fusion is triggered by high-affinity Ca²⁺ sensors. Because of their important roles in vesicle secretion, their Ca²⁺ binding ability, and their high expression levels in the brain (20, 31, 35–38), we addressed the roles of Doc2a/b, Syt1, and Syt7 in neuronal DCV fusion.In this study, we used primary Doc2a/b-, Syt1-, and Syt7-null (knockout, KO) neurons expressing DCV fusion reporters (34, 39–41) with single-vesicle resolution. We show that both Syt1 and Syt7, but not Doc2a/b, are required for ∼60 to 90% of DCV fusion events. Deficiency of both Syt1 and Syt7 did not produce an additive effect, suggesting they function in the same pathway. Syt1 overexpression (Syt1-OE) rescued DCV fusion in Syt7-null neurons, and vice versa, indicating that the two proteins compensate for each other in DCV secretion. Moreover, overexpression of Syt1 or Syt7 in wild-type (WT) neurons increased DCV fusion, suggesting they are both rate limiting for DCV secretion. We conclude that DCV fusion requires two calcium sensors, Syt1 and Syt7, that act in a single/serial pathway and that both sensors regulate fusion in a rate-limiting and dose-dependent manner.     

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H⁺ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H⁺ release, the H⁺ is not metastably trapped in such a putative H⁺ acceptor, unlike the other H⁺ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H⁺ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the β-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H⁺ release; H⁺ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H⁺ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H⁺ release mechanisms.

Microbial rhodopsins are a large family of heptahelical photoreceptive membrane proteins that use retinal as a chromophore (1). They are found in diverse microorganisms such as bacteria, archaea, algae, protists, fungi, and giant viruses (2–4). The retinal chromophore in the microbial rhodopsins undergoes all-trans to 13-cis isomerization upon light illumination, leading to a photocyclic reaction in which the proteins exert their various biological functions. Ion transporting rhodopsins are the most abundant microbial rhodopsins and are classified into light-driven ion pumps and light-gated ion channels. Whereas light-driven ion pumps actively transport ions in one direction, light-gated ion channels passively transport them according to the electrochemical potential. Ion transporting rhodopsins are used as important molecular tools in optogenetics to control neural firing in vivo. Microbial rhodopsins evolved independently from animal rhodopsins, which are also retinal-bound heptahelical proteins and a subgroup of G protein–coupled receptors. The third class of rhodopsin, heliorhodopsin (HeR), was recently reported (5–7). It has an inverted protein orientation in the membrane, as compared with microbial and animal rhodopsins (5).Bacteriorhodopsin (BR) is the first ion pump rhodopsin found in the haloarchaeon (8) Halobacterium salinarum, and it transports protons (H⁺) outward. An inward chloride (Cl⁻) pump, halorhodopsin, was subsequently identified in the same species (9, 10) (SI Appendix, Table S1). Although an outward sodium pump rhodopsin was not found for several decades after the discovery of BR, it was eventually identified in the marine bacterium Krokinobacter eikastus in 2013 (11). These ion-pumping rhodopsins hyperpolarize the membrane by their active ion transport against the electrochemical potential of the membrane. However, the bacterial xenorhodopsins (XeRs) reportedly work as light-driven inward H⁺ pumps (12). Thus, the membrane potentials are not exclusively hyperpolarized via active transport by ion pumping.Asgard archaea are the closest prokaryotic species to ancestral eukaryotes (13) and have many genes previously thought to be unique to eukaryotes. Recently, a new microbial rhodopsin group, schizorhodopsin (SzR), was found in the assembled genomes of Asgard archaea and the metagenomic sequences of unknown microbial species (14, 15) (SI Appendix, Table S1). A molecular phylogenetic analysis suggested that SzRs are located at an intermediate position between typical microbial rhodopsins, also called “type-1 rhodopsins” (16), and HeR (5). Thus they were named “schizo- (meaning “split” in Greek)” rhodopsin. Especially the transmembrane helix (TM) 3 of SzR is more similar to that of HeR than type 1. By contrast, TM6 and 7 of SzR and type-1 rhodopsins share many identical residues (e.g., W154, P158, W161, D184, and F191), which are not conserved in HeR (15). SzRs heterologously expressed in Escherichia coli and mammalian cells displayed light-driven inward H⁺ pump activity (15). As SzRs are phylogenetically distant from XeRs (∼18% identity and ∼44% similarity), these two rhodopsin families with similar functions are thought to have convergently evolved.In both SzR and XeR, an H⁺ is released from the Schiff base linkage connecting the retinal and a conserved lysine residue (retinal Schiff base, RSB) in TM7 to the cytoplasmic side. The transiently deprotonated RSB shows a largely blue-shifted absorption peak, and this blue-shifted state was named the M-intermediate. In the case of XeR from the marine bacterium Parvularcula oceani (PoXeR), the H⁺ is transferred to the cytoplasmic aspartate (PoXeR D216, H⁺ acceptor) in TM7 and then released to the cytoplasmic bulk phase (12). By contrast, the H⁺ acceptor of SzR was considered to be E81 in TM3 since the mutation of E81 to glutamine abolished the inward H⁺ transport (15). However, the H⁺ is not metastably trapped in E81, probably for a kinetic reason: The rate of H⁺ release from E81 to the cytoplasmic bulk phase might be faster than that of H⁺ transfer from RSB to E81. The reason why SzR and PoXeR exhibit different kinetic behaviors in H⁺ release has not been elucidated. Subsequently, another H⁺ is taken up from the extracellular side and directly transferred from the extracellular bulk phase to the RSB during the M-decay to the initial state.Recently, a new SzR subgroup, AntR, was identified in metagenomic data obtained from Antarctic freshwater lake samples (17). Although SzR and AntR share substantial similarities (identity: ∼33%; similarity: ∼56%), and most of the SzR residues essential for the inward H⁺ pump function are conserved in AntR (e.g., SzR R67, F70, C75, E81, D184, and K188), they have several differences. While the SzR E81Q mutant cannot transport H⁺, as mentioned above, the H⁺ transport efficiency of AntR E81Q is close to that of AntR wild type (WT) (17), suggesting the diversity of H⁺ transport mechanisms. To understand the inward H⁺ pump mechanism of SzR as well as the similarities and differences between SzR, XeR, and AntR we present a three-dimensional structure of an SzR.     

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH₃/NH₄⁺) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH₄⁺ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Ammonium transporters (AMTs), methylammonium permeases (MEPs), and Rhesus glycoproteins (Rh proteins) comprise a protein family with three clades, and homologs from each have been identified in virtually all domains of life (1). AMT proteins were first identified in plants (2) with the simultaneous discovery of MEP proteins in fungi (3), followed by Rh proteins in humans (4). Ammonia (NH₃/NH₄⁺) is vital for growth in plants and microorganisms and is retained in some animals for use as an osmolyte (5, 6), for buoyancy (7, 8), and for those lacking sufficient dietary nitrogen (9). In the majority of animals, however, ammonia is the toxic by-product of amino acid and nucleic acid metabolism and, accordingly, requires efficient mechanisms for its regulation, transport, and excretion (10–13). AMT, MEP, and Rh proteins are responsible for the selective movement of ammonia (NH₃) or ammonium (NH₄⁺) across biological membranes, a process that all organisms require. Unlike their vertebrate, bacterial, and fungal counterparts which function as putative NH₃ gas channels (14–18), a myriad of evidence suggests that plant AMT proteins and closely related members in some animals are functionally distinct and facilitate electrogenic ammonium (NH₄⁺) transport (17, 19–22). In contrast to vertebrates which only possess Rh proteins (23), many invertebrates are unique in that they express both AMT and Rh proteins, sometimes in the same cell (24–28). Among insects, the presence of both AMT and Rh proteins has been described in Drosophila melanogaster (29, 30) and mosquitoes that vector disease-causing pathogens, Anopheles gambiae (22, 31) and Aedes aegypti (32, 33). It is unclear whether, in these instances, AMT and Rh proteins can functionally substitute for one another, but in the anal papillae of A. aegypti larvae, knockdown of either Amt or Rh proteins causes decreases in ammonia transport, suggesting that they do not (32–34). To date, studies on ammonia transporter (AMT and Rh) function in insects have focused on ammonia sensing and tasting in sensory structures (22, 30, 31, 35), ammonia detoxification and acid–base balance in muscle, digestive, and excretory organs (15, 36), and ammonia excretion in a variety of organs involved in ion and water homeostasis (9, 24, 32–34).A. aegypti is the primary vector for the transmission of the human arboviral diseases Zika, yellow fever, chikungunya, and dengue virus, which are of global health concern due to rapid increases in the geographical distribution of this species, presently at its highest ever (37, 38). In light of the well-documented evolution of insecticide resistance in mosquitoes (39–42), more recent methods to control disease transmission such as the sterile insect technique (43), transinfection and sterilization of mosquitoes with the bacterium Wolbachia (44), and targeted genome editing rendering adult males sterile (45) have proven effective. These methods take advantage of various aspects of mosquito reproductive biology; however, an understanding of male reproductive biology and the male contributions to female reproductive processes is still in its infancy (46). Here, we describe the expression of an A. aegypti ammonium transporter (AeAmt1) in the sperm during all stages of spermatogenesis, spermiogenesis, and egg fertilization, which is critical for fertility.     

Underpotential lithium plating on graphite anodes caused by temperature heterogeneity

Rechargeability and operational safety of commercial lithium (Li)-ion batteries demand further improvement. Plating of metallic Li on graphite anodes is a critical reason for Li-ion battery capacity decay and short circuit. It is generally believed that Li plating is caused by the slow kinetics of graphite intercalation, but in this paper, we demonstrate that thermodynamics also serves a crucial role. We show that a nonuniform temperature distribution within the battery can make local plating of Li above 0 V vs. Li⁰/Li⁺ (room temperature) thermodynamically favorable. This phenomenon is caused by temperature-dependent shifts of the equilibrium potential of Li⁰/Li⁺. Supported by simulation results, we confirm the likelihood of this failure mechanism during commercial Li-ion battery operation, including both slow and fast charging conditions. This work furthers the understanding of nonuniform Li plating and will inspire future studies to prolong the cycling lifetime of Li-ion batteries.

Lithium (Li)-ion batteries with graphite anodes and Li metal oxide cathodes are the dominant commercial battery chemistry for electric vehicles (EVs) (1). However, their cycle lifetime and operational stability still demand further improvements (2–5). During long-term cycling, Li-ion batteries undergo irreversible capacity decay due to decreased utilization of anode/cathode active materials, metallic Li plating, electrolyte dry-out, impedance build-up, or excessive heat generation (6–9). Some of these issues also lead to battery shorting and thermal runaway (10, 11). To enable mass adoption of EVs, increasing efforts have been made to realize the fast charging of Li-ion batteries (12). Under this condition, all of the detrimental factors mentioned above are aggravated (6, 7, 13), further compromising the battery cycling life and safety. As a result, a clear understanding of the failure mechanisms of Li-ion batteries is crucial for their future development.Plating of metallic Li on graphite anodes is a major cause of the capacity decay of Li-ion batteries (6, 7, 12, 14–17). Significant amounts of solid electrolyte interphase (SEI) and dead Li form and remain inactive, leading to an accelerated loss of Li inventory. It is generally believed that the slow kinetics of Li ion intercalation into graphite causes metallic Li plating (14). Three-electrode measurements (18–25) showed that the potential of graphite anodes shifted negatively under increased charging rates and finally dropped below 0 V vs. Li⁰/Li⁺, reaching Li-plating conditions. However, Li-plating phenomenon on graphite anodes is still not fully understood. Firstly, the actual onset potential of Li plating is still unclear, which is not necessarily below 0 V vs. Li⁰/Li⁺ (18). Furthermore, few studies explained why Li plated on graphite in spatially inhomogeneous patterns (7, 14, 17). Most importantly, in some reports, Li plates even under a moderate charging rate below 1.5 C (6, 7). Under these conditions, three-electrode measurements indicate that the anode potential does not drop below 0 V vs. Li⁰/Li⁺ (18). Kinetic arguments alone are not sufficient to resolve these problems, so we hypothesize that previously neglected thermodynamic factors may also play crucial roles in Li plating.It is well-known that the equilibrium electrode potential of a redox reaction shifts with temperature (26–35). Exothermic reactions and joule heating during cycling raise the temperature of batteries (10), which can also build up an internal temperature gradient. Simulations (7, 36–42) and experimental studies (41, 43–49) showed intensified heating under increased cycling rates, and temperature differences of 2 K to nearly 30 K within the batteries (10). This spatial variation in temperature leads to a heterogeneous distribution of the equilibrium potential for both Li plating and graphite intercalation on the anode, which could make Li plating thermodynamically favorable at certain locations.In this paper, we discover that temperature heterogeneities within Li-ion batteries can cause Li plating by shifting its equilibrium electrode potential. We first introduce a method to quantify the temperature dependence of the equilibrium potential for both Li plating and graphite intercalation. Then, we correlate the shift of the equilibrium potential to Li plating using a Li-graphite coin cell with an intentionally created heterogeneous temperature distribution and explain the observation with thermal and electrochemical simulations. Finally, the effects under fast charging conditions are examined. The data explicitly show that metallic Li can plate above 0 V vs. Li⁰/Li⁺ (room temperature) on a graphite anode. The temperature dependence of the equilibrium potential likely participates in the capacity decay of commercial Li-ion batteries, which can be increasingly severe during fast charging conditions. This research brings insights into a key failure mechanism of Li-ion batteries, highlights the importance of maintaining homogeneous temperature within batteries, and will inspire future development of Li-ion batteries with improved safety and cycle lifetime.