α-Neurotoxins, a prominent class of snake toxins belonging to the three-finger toxin (3FTx) superfamily, function as orthosteric antagonists of postsynaptic nicotinic acetylcholine receptors (nAChRs). Due to their high selectivity, α-neurotoxins have been widely used to characterize these physiologically important receptors. In this study, we investigated the concentration-response relationships of wild-type erabutoxin A (Ea), an α-neurotoxin, and six of its single-residue mutants by assessing their ability to inhibit acetylcholine-evoked currents mediated by α1β1γδ (fetal) and α1β1γε (adult) isoforms of human muscle-type nAChRs. These receptors were heterologously expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp electrophysiology. Our results revealed that two mutants exhibited selectivity for the α1-δ interface: the S8T mutant displayed a selectivity pattern of α1-δ > α1-γ > α1-ε, whereas the K47E mutant showed α1-δ ≥ α1-γ > α1-ε. In contrast, the K27E and D31H mutants preferentially targeted the α1-ε interface (α1-ε > α1-γ ≥ α1-δ). The Q10A mutant showed no interface selectivity, and the R33E mutant largely lost its antagonist activity. These findings allowed us to define interface selectivity based on interactions between the mutants and six structural loops of the nAChR. This functional characterization enhances our understanding of receptor subtype selectivity and supports the future design of functionally selective protein probes targeting nAChRs and other ligand-gated ion channels.

Modulation of voltage-dependent calcium and potassium channels by G protein-coupled receptors (GPCRs) plays a key role in reducing nociceptive transmission. Specifically, baclofen and the analgesic peptide α-conotoxin Vc1.1 activate GABAB receptors, resulting in the inhibition of CaV2.2 and CaV2.3 calcium channels, as well as the potentiation of GIRK1/2 potassium channels in mammalian primary afferent neurons. In this study, we examined the expression of these key ion channel targets in rodent and human dorsal root ganglion (DRG) neurons. We examined how CaV2.2 and GIRK channel antagonists, as well as a GIRK channel activator, influence the passive and active electrical properties of adult mouse DRG neurons. Additionally, we assessed the effects of α-conotoxin Vc1.1 on neuronal excitability in the presence of the selective CaV2.2 antagonist ω-conotoxin CVIE and the GIRK channel activator ML297. Furthermore, we evaluated how the GIRK channel antagonist Tertiapin-Q affects the excitability of mouse colonic DRGs and colonic afferents and explored the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in regulating the membrane excitability of colonic DRGs. Our findings suggest that both CaV2.2 inhibition and GIRK channel potentiation contribute to the reduction of neuronal excitability in mouse DRGs, mediating the analgesic effects of Vc1.1 and baclofen observed in vivo. However, our findings indicate that GIRK channel potentiation may have a limited role in the mechanisms underlying Vc1.1 and baclofen action in colon-innervating DRGs and colonic afferents. ### Competing Interest Statement The authors have declared no competing interest.

Parkinson's disease (PD) is a complex, progressive neurodegenerative disorder driven by multiple pathogenetic factors, including oxidative stress, mitochondria dysfunction, neuroinflammation, and ion imbalance. Recent evidence highlights the significant role of potassium channels in the pathophysiology of PD. We recently identified a PD-linked genetic mutation in the KCNJ15 gene (KCNJ15p.R28C), encoding the inwardly rectifying potassium channel Kir4.2, within a four-generation family with familial PD. However, the role of the Kir4.2 channel in neurodegenerative diseases remains largely unexplored. This study aimed to elucidate the impact of the KCNJ15p.R28C (Kir4.2R28C) mutation on the biophysical and biochemical properties of Kir4.2. Employing Kir4.2-overexpressing HEK293T cells as a model, we investigated how the mutation affects the channel's functional properties, total protein expression, intracellular processing in the endoplasmic reticulum and lysosomes, and plasma membrane trafficking. Patch clamp studies revealed that the Kir4.2R28C mutation results in loss of channel function with significant dominant-negative effects. This dysfunction is partially attributed to the substantial reduction in overall mutant channel protein expression compared to the wild-type (Kir4.2WT). We observed that both Kir4.2WT and Kir4.2R28C proteins undergo glycosylation during the post-translational modification process, albeit with differing protein turnover efficiencies. Furthermore, the Kir4.2R28C mutant exhibits reduced stability and compromised plasma membrane trafficking capacity compared to Kir4.2WT. These findings suggest that the Kir4.2R28C mutant has unique biomolecular and biophysical characteristics distinct from the Kir4.2WT channel, which potentially elucidates its role in the pathogenesis of PD.

Natural toxins are highly effective at targeting ion channels with high selectivity and potency. To date, all identified spider venom peptide toxins that modulate voltage-gated potassium (KV) channels inhibit Shab (KV2) or Shal-related isoforms (KV4) by interacting with their voltage-sensing domains. In this study, we report novel spider-derived pore-blocking toxins that selectively target Shaker-type (KV1) channels with nanomolar potency. We isolated murinotoxins MnTx-1 and MnTx-2 from the orange baboon tarantula Pterinochilus murinus and sequenced them using a combination of Edman degradation, mass spectrometry, and venom gland nanopore transcriptomics. MnTx-1 was produced recombinantly, and its NMR solution structure was determined. Although MnTx-1 shares sequence motifs common to spider toxins, it displays a distinctly different three-dimensional structure, featuring an alternative disulfide linkage, which we have termed the Disulfide-Reined Hairpin (DRH). We attribute the unique pharmacology of MnTx-1 to its unusual spatial structure. The DRH motif represents a promising new miniature scaffold for future bioengineering applications.

ABSTRACTGABAB receptor (GABABR) activation is known to alleviate pain by reducing neuronal excitability, primarily through inhibition of high voltage‐activated (HVA) calcium (CaV2.2) channels and potentiating G protein–coupled inwardly rectifying potassium (GIRK) channels. Although the analgesic properties of small molecules and peptides have been primarily tested on isolated murine dorsal root ganglion (DRG) neurons, emerging strategies to develop, study, and characterise human pluripotent stem cell (hPSC)‐derived sensory neurons present a promising alternative. In this study, hPSCs were efficiently differentiated into peripheral DRG‐induced sensory neurons (iSNs) using a combined chemical and transcription factor‐driven approach via a neural crest cell intermediate. Molecular characterisation and transcriptomic analysis confirmed the expression of key DRG markers such as BRN3A, ISLET1, and PRPH, in addition to GABABR and ion channels including CaV2.2 and GIRK1 in iSNs. Functional characterisation of GABABR was conducted using whole‐cell patch clamp electrophysiology, assessing neuronal excitability under current‐clamp conditions in the absence and presence of GABABR agonists baclofen and α‐conotoxin Vc1.1. Both baclofen (100 μM) and Vc1.1 (1 μM) significantly reduced membrane excitability by hyperpolarising the resting membrane potential and increasing the rheobase for action potential firing. In voltage‐clamp mode, baclofen and Vc1.1 inhibited HVA Ca2+ channel currents, which were attenuated by the selective GABABR antagonist CGP 55845. However, modulation of GIRK channels by GABABRs was not observed in the presence of baclofen or Vc1.1, suggesting that functional GIRK1/2 channels were not coupled to GABABRs in hPSC‐derived iSNs. This study is the first to report GABABR modulation of membrane excitability in iSNs by baclofen and Vc1.1, highlighting their potential as a future model for studying analgesic compounds.image

α-Conotoxin Vc1.1 is a disulfide-rich peptide and a promising drug candidate for treating neuropathic and chronic pain. Backbone cyclization was applied to enhance its drug-like properties, resulting in improved serum stability and oral bioavailability. However, this modification also adversely affected its stability and activity in simulated intestinal fluid (SIF). To address these adverse effects, we explored the use of polyethylene glycol (PEG) linkers as substitutes for peptide backbone cyclization linkers. PEG linkers are smaller, more flexible, and more stable than peptide linkers. Furthermore, previous studies have demonstrated that PEG backbone linkers can enhance the activity of conotoxins. In this study, we synthesized four PEG-backboned cyclic Vc1.1 (cVc1.1) analogues with varying lengths of PEG linkers and used a chemo-enzymatic method to cyclize these analogues. Their structure, stability, and activity were subsequently evaluated. Although the results revealed that PEG linkers preserved the SIF stability and activity of cVc1.1, they highlighted the crucial role of the peptide's helical structure in maintaining its stability and activity. Additionally, this work introduces a novel approach for synthesizing cyclic conotoxins.

Sensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.### Competing Interest StatementThe authors have declared no competing interest.

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain.

α-conotoxins (α-Ctxs), a class of disulfide-rich conopetides, are excellent drug leads due to their small size, high selectivity, and potency for specific membrane receptors and ion channels involved in pain transmission. However, their high susceptibility to proteolytic degradation limits their therapeutic potential. In this study, we designed and synthesized a series of conformationally stable analogues of α-Ctx Mr1.1[S4Dap] using various structural optimization strategies. The Mr1.1[S4Dap, C16Pen] analogue maintained potency at human α9α10 nicotinic acetylcholine receptors, with a half-maximal inhibitory concentration (IC50) of 4 nM. It exhibited over a 5-fold increase in serum stability compared to Mr1.1[S4Dap], without disrupting its overall conformation. Furthermore, intravenous application of Mr1.1[S4Dap, C16Pen] showed potent analgesic activity in oxaliplatin-induced cold allodynia, indicating a high potential for drug development. Overall, the results from this study provide valuable insights for optimizing the serum stability of disulfide-rich peptides in future therapeutic applications.

α9α10 nicotinic acetylcholine receptors (nAChRs) are a promising nonopioid analgesic target, with α9α10 nAChR antagonists showing efficacy against chemotherapy-induced hyperalgesia and allodynia. GeX-2, a potent analgesic conotoxin antagonist of α9α10 nAChRs, has limited serum stability. This study improved GeX-2 stability by capping its N-terminal with fatty acids or polyethylene glycol chains, which enhanced its serum stability but eliminated activity at G protein-coupled γ-aminobutyric acid type B (GABAB) receptor-coupled CaV2.2 channels while preserving activity at α9α10 nAChRs. In vivo, α9α10 nAChRs antagonism alone did not alleviate neuropathic pain, highlighting the importance of GABAB receptor-coupled CaV2.2 channels in GeX-2's antinociceptive effects in the chronic constriction injury rat model. The GeX-2 analogue, with an N-terminal methyl group, showed improved activity and selectivity for α9α10 nAChRs, increased serum half-life, and strong analgesic effects in oxaliplatin-induced cold allodynia models. AlphaFold3 and molecular dynamics simulations provided insights into the binding modes and the effects of N-terminal capping, which informed future peptide therapeutic developments.

The α-conotoxins (α-Ctxs) are short, disulfide-rich peptides derived from the venom of the Conus marine snails, primarily acting as antagonists of nicotinic acetylcholine receptors (nAChRs). Specifically, α-Ctx Vc1.1, a 16-amino acid peptide from Conus victoriae, competitively antagonizes non-muscle nAChRs, inhibits nicotine-induced currents in bovine chromaffin cells, and alleviates neuropathic pain in rat models. Although Vc1.1 selectively inhibits rat α9α10 nAChRs, its potency and selectivity across human nAChR subtypes remain unresolved. In this study, we assessed the activity of Vc1.1 on human (h) nAChRs heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique and simulated interactions using computational modeling. Vc1.1 selectively antagonized homomeric α9 and heteromeric α3β2 nAChRs, with half-maximal inhibitory concentrations (IC50) of 160 nM and 232 nM, respectively. At hα9[N179A]α10, Vc1.1 exhibited a 20-fold decrease in potency compared to hα9α10, due to the loss of hydrogen bonding with Vc1.1-D11. Conversely, Vc1.1 was four-fold more potent at hα3β2[E86A] compared to hα3β2, possibly influenced by the proximal residue β2-K104, as suggested by molecular dynamics (MD) simulations. Additionally, Vc1.1's potency doubled at hα9[N213K]α10, whereas it remained unchanged at hα9[N213R]α10 nAChRs. MD simulations indicate that altered interactions between the mutant hα9 N179A, N213K, and N213R side chains and Vc1.1-D5 may partly explain these changes in potency. The inhibitory action of Vc1.1 at α9-containing nAChRs is particularly relevant given their role in neuroinflammation, presenting a potential therapeutic pathway for alleviating neuropathic and inflammatory pain. This study provides valuable insights into the rational design of Vc1.1-derived α-Ctxs with enhanced nAChR subtype selectivity.

Pain severely affects the physical and mental health of patients. The need to develop nonopioid analgesic drugs to meet medical demands is urgent. In this study, we designed a truncated analogue of alpha O-conotoxin, named GeX-2, based on disulfide-bond deletion and sequence truncation. GeX-2 retained the potency of its parent peptide at the human alpha 9 alpha 10 nAChR and exhibited potent inhibitory activity at Ca(V)2.2 channels via activation of the GABA(B) receptor (GABA(B)R). Importantly, GeX-2 significantly alleviated pain in the rat model of chronic constriction injury. The dual inhibition of GeX-2 at both alpha 9 alpha 10 nAChRs and Ca(V)2.2 channels is speculated to synergistically mediate the potent analgesic effects. Results from site-directed mutagenesis assay and computational modeling suggest that GeX-2 preferentially interacts with the alpha 10(+)alpha 10(-) binding site of alpha 9 alpha 10 nAChR and favorably binds to the top region of the GABA(B)R2 subunit. The study offers vital insights into the molecular action mechanism of GeX-2, demonstrating its potential as a novel nonopioid analgesic.

Wnt signalling must be 'just right' to promote tumour growth. Basal cell adenoma (BCA) and basal cell adenocarcinoma (BCAC) of the salivary gland are rare tumours that can be difficult to distinguish from each other and other salivary gland tumour subtypes. Due to their rarity, the genetic profiles of BCA and BCAC have not been extensively explored. Using whole-exome and transcriptome sequencing of BCA and BCAC cohorts, we identify a novel recurrent FBXW11 missense mutation (p.F517S) in BCA, that was mutually exclusive with the previously reported CTNNB1 p.I35T gain-of-function (GoF) mutation. These driver events collectively accounted for 94% of BCAs. In vitro , mutant FBXW11 had a dominant negative affect, characterised by defective binding to β-catenin and the accumulation of β-catenin in cells. This was consistent with the nuclear expression of β-catenin observed in BCA cases harbouring the FBXW11 p.F517S mutation and activation of the Wnt/β-catenin pathway. The genomic profiles of BCAC were distinct from BCA, with hotspot DICER1 and HRAS mutations and putative driver mutations affecting PI3K/AKT and NF-κB signalling pathway genes. A single BCAC, which may represent a malignant transformation of BCA, harboured the recurrent FBXW11 mutation. These findings have important implications for the diagnosis and treatment of BCA and BCAC, which, despite histopathologic overlap, may be unrelated entities. ### Competing Interest Statement The authors have declared no competing interest.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the r alpha 1 beta 2 gamma 2L gamma- aminobutyric acid type A receptor (GABA(A) R), and the human voltage-gated N -type calcium channel (Ca(V)2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: alpha 9 alpha 10 > alpha 7 > alpha 3 beta 2 congruent to alpha 3 beta 4, indicating that beta 2/ beta 4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at h alpha 7 and h alpha 9 alpha 10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of r alpha 7 was ACh concentrationindependent and voltage-dependent, whereas r alpha 9 alpha 10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the alpha 7 ion channel pore and alpha 9 alpha 10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the alpha 7 model TBG forms stable interactions with luminal rings at 9 ' , 13 ' , and 16 ' , whereas IBG mostly interacts with the extracellular-transmembrane junction. In the alpha 9 alpha 10 model, however, these compounds interacted with several residues from the principal ( +) and complementary (-) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at alpha 7, and one alpha 9 alpha 10 orthosteric site. TBG and IBG inhibited the GABA(A) R and Ca(V)2.2 channels with 10 to 30 -fold lower potencies. In sum, we show that TBG and IBG inhibit the alpha 7 and alpha 9 alpha 10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABA(A) R and Ca(V)2.2 channel.

Live-cell imaging can reveal dynamic and multimodal cell signaling by monitoring calcium flux. Spatiotemporal changes in Ca2+ concentrations instigate specific downstream processes and by categorizing these events, we can examine the language cells use to communicate both to themselves and with each other. Thus, calcium imaging is an understandably popular and versatile technique that relies on high-resolution optical data as measured by fluorescence intensity. This is executed with relative ease on adherent cells, as changes in fluorescence intensity can be monitored over time in fixed regions of interest. However, perfusion of non-adherent or mildly adherent cells leads to their mechanical displacement thereby hindering the spatial resolution of fluorescence intensity changes through time. Here we provide details of a simple and cost-effective protocol using gelatin to prevent cell dislodgement during the solution exchanges that occur during recording.

Supplementary Data from A High-Throughput Pharmaceutical Screen Identifies Compounds with Specific Toxicity against BRCA2-Deficient Tumors