Introduction Antimicrobial peptides (AMPs) hold promises to counter antimicrobial resistance. They cause several deleterious effects in bacterial cells by interfering with cell wall biosynthesis, DNA replication, transcription, and translation. Objectives The essential role of cellular respiration in bacterial survival underscores its potential as a target for antimicrobial intervention. However, few studies have explored if and how AMPs target bacterial respiratory terminal oxidases, which contribute to proton motive force (PMF) for ATP synthesis and are essential for cell survival. Methods In vitro and in vivo experiments are performed to examine the antimicrobial activities of hc-cath, the first sea snake-derived cathelicidin peptide, against A. baumannii. Mode of action (MOA) of hc-cath is confirmed through a series of biochemical, enzymatic, and biophysical assays along with transcriptome analysis. Results In this investigation, hc-cath is found to interact with and inhibit the terminal oxidase bo3 in A. baumannii, disrupting PMF and inducing toxic reactive oxygen species (ROS) production. Mitigating oxidative stress rescues bacterial death induced by hc-cath. Phenotypic validation using a cyoB (encoding oxidase bo3)-deficient strain further confirms the reduced peptide susceptibility and attenuates respiratory metabolic perturbation. Conclusion Our research identifies oxidase bo3, which predominates under high aeration growth conditions, as a key potential target of AMPs, underscoring bacterial respiratory metabolism as a viable therapeutic focus for AMPs-based strategies.

Plants have evolved diverse strategies to defend against herbivores, including structural barriers such as trichomes and tough leaves, the production of toxic secondary metabolites, the emission of volatile organic compounds (VOCs), and the recruitment of natural predators to deter herbivory. However, the molecular mechanisms underlying their ability to deter large herbivorous mammals remain poorly understood. In this study, we demonstrate that the order Piperales, which is particularly rich in VOCs, employs a conserved chemical defense strategy targeting herbivorous mammals. Behavioral assays, transgenic models, and electrophysiological analyses revealed that VOCs from Piperales species, particularly Houttuynia cordata, activate TRPA1-a sensory ion channel critical for detecting irritants-in mice and herbivores such as cattle and goats. A stable derivative of the key VOC houttuynin, sodium houttuyfonate (SH), selectively activated TRPA1 by binding conserved cysteine residues, triggering aversion in herbivorous mammals. Crucially, TRPA1 activation sites upon VOC application were conserved across herbivorous species, suggesting that Piperales plants employ a conserved evolutionary strategy to defend against herbivorous mammals. Our findings reveal a compelling case of lineage-specific defensive adaptation within Piperales, providing novel insights into plant-herbivore interactions. This research deepens our understanding of the critical role of chemical defenses in plant survival, adaptation, and ecological niche specialization.

The development of noninvasive approaches to precisely control neural activity in mammals is highly desirable. Here, we used the ion channel transient receptor potential ankyrin-repeat 1 (TRPA1) as a proof of principle, demonstrating remote near-infrared (NIR) activation of endogenous neuronal channels in mice through an engineered nanoagonist. This achievement enables specific neurostimulation in nongenetically modified mice. Initially, target-based screening identified flavins as photopharmacological agonists, allowing for the photoactivation of TRPA1 in sensory neurons upon ultraviolet A/blue light illumination. Subsequently, upconversion nanoparticles (UCNPs) were customized with an emission spectrum aligned to flavin absorption and conjugated with flavin adenine dinucleotide, creating a nanoagonist capable of NIR activation of TRPA1. Following the intrathecal injection of the nanoagonist, noninvasive NIR stimulation allows precise bidirectional control of nociception in mice through remote activation of spinal TRPA1. This study demonstrates a noninvasive NIR neurostimulation method with the potential for adaptation to various endogenous ion channels and neural processes by combining photochemical toolboxes with customized UCNPs.

Numerous missense mutations in cyclic nucleotide-gated (CNG) channels cause achromatopsia and retinitis pigmentosa, but the underlying pathogenic mechanisms are often unclear. We investigated the structural basis and molecular/cellular effects of R410W, an achromatopsia-associated, presumed loss-of-function mutation in human CNGA3. Cryo-EM structures of the Caenorhabditis elegans TAX-4 CNG channel carrying the analogous mutation, R421W, show that most apo channels are open. R421, located in the gating ring, interacts with the S4 segment in the closed state. R421W disrupts this interaction, destabilizes the closed state, and stabilizes the open state. CNGA3_R410W/CNGB3 and TAX4_R421W channels are spontaneously active without cGMP and induce cell death, suggesting cone degeneration triggered by spontaneous CNG channel activity as a possible cause of achromatopsia. Our study sheds new light on CNG channel allosteric gating, provides an impetus for a reevaluation of reported loss-of-function CNG channel missense disease mutations, and has implications for mutation-specific treatment of retinopathy.

Cyclovirobuxine D (CVB-D), the main active constituent of traditional Chinese medicine Buxus microphylla, was developed as a safe and effective cardiovascular drug in China. B. microphylla has also been used to relieve various pain symptoms for centuries. In this study, we examined and uncovered strong and persistent analgesic effects of cyclovirobuxine D against several mouse models of pain, including carrageenan- and CFA-induced inflammatory pain and paclitaxel-mediated neuropathic hypersensitivity. Cyclovirobuxine D shows comparable analgesic effects by intraplantar or intraperitoneal administration. Cyclovirobuxine D potently inhibits voltage-gated Cav2.2 and Cav3.2 channels but has negligible effects on a diverse group of nociceptive ion channels distributed in primary afferent neurons, including Nav1.7, Nav1.8, TRPV1, TPRA1, TRPM8, ASIC3, P2X2 and P2X4. Moreover, inhibition of Cav3.2, rather than Cav2.2, plays a dominant role in attenuating the excitability of isolated dorsal root ganglion neurons and pain relieving effects of cyclovirobuxine D. Our work reveals that a currently in-use cardiovascular drug has strong analgesic effects mainly via blockade of Cav3.2 and provides a compelling rationale and foundation for conducting clinical studies to repurpose cyclovirobuxine D in pain management.

Cyclic nucleotide–gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP−activated channel TAX-4, reconstituted in lipid nanodiscs. These structures, together with computational and functional analyses and a mutant channel structure, reveal a double-barrier hydrophobic gate formed by two S6 amino acids in the central cavity. cGMP binding produces global conformational changes that open the cavity gate located ~52 Å away but do not alter the structure of the selectivity filter—the commonly presumed activation gate. Our work provides mechanistic insights into the allosteric gating and regulation of CN-gated and nucleotide-modulated channels and CNG channel−related channelopathies. Cryo-EM structures of a C. elegans cGMP-activated channel TAX-4 in lipid nanodiscs reveal a hydrophobic gate in the central cavity and, together with electrophysiology, provide mechanistic insights into the gating and regulation of CNG channels.

Animal toxins that are used to subdue prey and deter predators act as the key drivers in natural food chains and ecosystems. However, the predators of venomous animals may exploit feeding adaptation strategies to overcome toxins their prey produce. Much remains unknown about the genetic and molecular game process in the toxin-dominant food chain model. Here, we show an evolutionary strategy in different trophic levels of scorpion-eating amphibians, scorpions and insects, representing each predation relationship in habitats dominated by the paralytic toxins of scorpions. For scorpions preying on insects, we found that the scorpion α-toxins irreversibly activate the skeletal muscle sodium channel of their prey（insect, Bg Na V1） through a membrane delivery mechanism and an efficient binding with the Asp/Lys-Tyr motif of Bg Na V1. However, in the predatory game between frogs and scorpions, with a single point mutation（Lys to Glu） in this motif of the frog’s skeletal muscle sodium channel（fNaV1.4）, fNaV1.4 breaks this interaction and diminishes muscular toxicity to the frog; thus, frogs can regularly prey on scorpions without showing paralysis. Interestingly, this molecular strategy also has been employed by some other scorpion-eating amphibians, especially anurans. In contrast to these amphibians, the Asp/Lys-Tyr motifs are structurally and functionally conserved in other animals that do not prey on scorpions. Together, our findings elucidate the protein-protein interacting mechanism of a toxin-dominant predator-prey system,implying the evolutionary game theory at a molecular level.

The mucolipin transient receptor potential (TRP) channels (TRPML1-3) localize primarily in endosomes and lysosomes. They conduct Ca2+ and Na+ currents from the lumen to the cytoplasm and play an important role in membrane trafficking, autophagy, exocytosis and ion homeostasis. Mutations of TRPML1 cause mucolipidosis type IV, a severe lysosomal storage disorder with cognitive, linguistic, visual and motor deficits. Dysfunction of TRPML3 causes deafness and pigmentation defects in mice. These severe effects underscore the crucial functional importance of TRPML channels. The activities of TRPML1 and TRPML3 are differentially regulated by the low endolysosomal pH. Thus, in the presence of Ca2+, TRPML1 currents are greatly enhanced by H+ whereas TRPML3 currents are inhibited by H+. To better understand the molecular mechanisms of TRPML channel function and regulation, we determined the structures of full length human TRPML3 in the apo, ML-SA1-bound, and low-pH-inhibited states by using cryoelectron microscopy, with resolutions of 4.06, 3.62 and 4.65 Å, respectively. The agonist ML-SA1 binds between S5 and S6 and opens an S6 gate. The selectivity filter is lined by a combination of carboxylate side-chains and backbone carbonyls and does not change significantly between the closed and open states. A polycystin-mucolipin domain forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore-loop, which undergoes dramatic conformational changes in response to low luminal pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with electrophysiological studies, reveal a new mechanism thereby luminal pH and other physiological modulators such as PIP2 regulate TRPML3 by changing S1 and S2 conformations.

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EM analyses confirmed that this architecture occurs in the full-length channel. Structure-function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.

The thiazolidine ring is a biologically active chemical structure and is associated with many pharmacological activities. However, the biological molecules that can interact with the thiazolidine ring are not known. We show that thiazolidine causes sustained activation of the TRPA1 channel and chemically reacts with glutathione, and the chemical reactivity of thiazolidine ring is required for TRPA1 activation. Reducing agents reverse thiazolidine-induced TRPA1 activation, and mutagenesis studies show that nucleophilic cysteine residues in TRPA1 are critical, suggesting an activation mechanism involving thioreactive chemical reactions. In vivo studies show that thiazolidine induces acute pain and inflammation in mouse and these responses are specifically dependent on TRPA1. These results indicate that thiazolidine compounds can chemically react with biological molecules containing nucleophilic cysteines, thereby exerting biological activities.

Cryo-EM analyses of human TRPML3 reveal this channel in three different states—closed, agonist-activated and low-pH-inhibited—and suggest mechanisms for regulation. TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is inhibited by low endolysosomal pH. Here we present cryo-electron microscopy (cryo-EM) structures of human TRPML3 in the closed, agonist-activated, and low-pH-inhibited states, with resolutions of 4.06, 3.62, and 4.65 Å, respectively. The agonist ML-SA1 lodges between S5 and S6 and opens an S6 gate. A polycystin-mucolipin domain (PMD) forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore loop, which undergoes dramatic conformational changes in response to low pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with the results of electrophysiological studies, indicate a new mechanism by which luminal pH and other physiological modulators such as PIP2 regulate TRPML3 by changing S1 and S2 conformations.

Bingpian is a time-honored herb in traditional Chinese medicine (TCM). It is an almost pure chemical with a chemical composition of (+)-borneol and has been historically used as a topical analgesic for millennia. However, the clinical efficacy of topical borneol lacks stringent evidence-based clinical studies and verifiable scientific mechanism. We examined the analgesic efficacy of topical borneol in a randomized, double-blind, placebo-controlled clinical study involving 122 patients with postoperative pain. Topical application of borneol led to significantly greater pain relief than placebo did. Using mouse models of pain, we identified the TRPM8 channel as a molecular target of borneol and showed that topical borneol-induced analgesia was almost exclusively mediated by TRPM8, and involved a downstream glutamatergic mechanism in the spinal cord. Investigation of the actions of topical borneol and menthol revealed mechanistic differences between borneol- and menthol-induced analgesia and indicated that borneol exhibits advantages over menthol as a topical analgesic. Our work demonstrates that borneol, which is currently approved by the US FDA to be used only as a flavoring substance or adjuvant in food, is an effective topical pain reliever in humans and reveals a key part of the molecular mechanism underlying its analgesic effect.

Iodine antiseptics exhibit superior antimicrobial efficacy and do not cause acquired microbial resistance. However, they are under-used in comparison with antibiotics in infection treatments, partly because of their adverse effects such as pain and allergy. The cause of these noxious effects is not fully understood, and no specific molecular targets or mechanisms have been discovered. In this study, we show that iodine antiseptics cause pain and promote allergic contact dermatitis in mouse models, and iodine stimulates a subset of sensory neurons that express TRPA1 and TRPV1 channels. In vivo pharmacological inhibition or genetic ablation of these channels indicates that TRPA1 plays a major role in iodine anti-septics- induced pain and the adjuvant effect of iodine antiseptics on allergic contact dermatitis and that TRPV1 is also involved. We further demonstrate that iodine activates TRPA1 through a redox mechanism but has no direct effects on TRPV1. Our study improves the understanding of the adverse effects of iodine antiseptics and suggests a means to minimize their side effects through local inhibition of TRPA1 and TRPV1 channels.