Stephan Alexander Pless

Ion channels remain notoriously recalcitrant drug targets despite their extensive pathophysiological significance. Acid-sensing ion channels (ASICs) are proton-gated cation channels that cause neuronal death when excessively activated, such as during ischaemic stroke. This acidosis-driven injury pathway remains unchallenged by existing therapeutics. Here we generate two de novo designed hASIC1a-specific miniproteins by developing a platform that integrates computational miniprotein design, high-throughput automated patch clamp screening, biophysical and structural characterisation, and in vivo validation. We identify a subtype-selective hASIC1a inhibitor, Denasin1, which achieves near-complete inhibition with nanomolar potency. Denasin1 is disordered in solution but adopts the computationally predicted helix-turn-helix fold when bound to the extracellular acidic pocket of hASIC1a. Crucially, Denasin1 reduces infarct volume by > 50% in a murine model of ischaemic stroke. We thereby establish our de novo design platform as a route to develop potent and selective ion channel modulators.

Dynamic conformational changes in intracellular domains fundamentally affect the function and pharmacology of many membrane proteins. For example, sodium influx through the cardiac voltage-gated sodium channel (NaV1.5) is rapidly terminated through conformational changes that result in pore closure, a transition known as inactivation. Inactivation involves NaV1.5 intracellular regions, particularly the DIII-DIV linker containing the IFM particle (Isoleucine-Phenylalanine-Methionine) and its dysfunction is a major cause of cardiac arrythmias. However, the conformational changes involved in inactivation and their modulation by auxiliary proteins and clinically used drugs remain incompletely characterized, partly because live-cell, site-specific labeling of intracellular regions with small fluorescent dyes remains challenging. Here, we combine live-cell protein semi-synthesis with voltage-clamp fluorometry (VCF), to track intracellular conformational dynamics of the cardiac sodium channel NaV1.5 and monitor their voltage dependence and kinetics in real time. We identify intracellular conformational changes involved in both fast and steady-state inactivation of NaV1.5 and show that both lidocaine and auxiliary proteins affect the kinetics of conformational changes of the DIII-DIV linker. Our work establishes the combination of protein semi-synthesis and voltage-clamp fluorometry as a powerful approach to dissect intracellular conformational changes in membrane proteins.

Acid-sensing ion channels (ASICs) are typically activated by acidic environments and contribute to nociception and synaptic plasticity. ASIC1a is the most abundant subunit in the central nervous system and forms homomeric channels permeable to Na ⁺ and Ca ²⁺ , making it a compelling therapeutic target for acidotic pathologies including stroke and traumatic brain injury. However, a complete conformational library of human ASIC1a has yet to be described. Here we show that human ASIC1a adopts six major conformations, resolved by cryo-electron microscopy across a pH range between 8.5 and 5.7 and in the presence of a toxin agonist and a gating-modifying amino acid substitution. These major conformations establish linear transmembrane helices to be associated with an open state, delineate mechanistic differences between proton and toxin activation and demonstrate that desensitization involves unexpected conformational diversity in the transmembrane domain. Together, they provide a three-dimensional framework to integrate previous structure–function studies on ASIC.

Neuronal resting membrane potential (RMP) reflects the balance of leak conductances and varies systematically across cortical cell types. Many GABAergic interneurons exhibit more depolarized RMPs than neighbouring excitatory neurons, but the molecular mechanisms underlying these systematic differences in intrinsic excitability remain incompletely defined. The sodium leak channel non-selective (NALCN) mediates a major fraction of basal sodium conductance and cause neurodevelopmental syndromes characterized by developmental delay and cognitive impairment. Here we define NALCN contribution to cortical circuit development and function with cell-type precision. Using Nalcn -GFP reporter line, we map Nalcn expression across cortical types revealing enrichment in GABAergic hippocampal interneurons relative to hippocampal pyramidal neurons. Using conditional mouse models, we selectively deleted Nalcn in cortical glutamatergic lineages or forebrain GABAergic cells from early embryogenesis. Electrophysiological analysis show that developmental loss of Nalcn preferentially reduces intrinsic excitability across GABAergic interneuron subtypes while sparing pyramidal neurons. To understand the impact of a depolarized GABAergic RMP on brain function, we assessed the behavioral performance of Nalcn -deficient mice that revealed persistent deficits in contextual adaptation and spatial short-term memory. These findings reveal a neuron type-specific function of NALCN in the cerebral cortex and position NALCN as a crucial ion channel regulating basal excitability of GABAergic inhibitory circuits and cortical circuit function.

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well‐structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point‐in‐time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70231 . The Concise Guides provide expert‐curated recommendations of ‘Gold Standard’ selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid‐2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

The sodium leak channel nonselective (NALCN) is the pore-forming channel component of the NALCN channelosome. This protein complex generates a depolarizing sodium leak in various tissues and contributes to respiratory and circadian rhythms, locomotion, and sleep. De novo and inherited variants of NALCN can lead to severe developmental syndromes called ‘contractures of the limbs and face, hypotonia, and developmental delay’ (CLIFAHDD) and ‘infantile hypotonia with psychomotor retardation and characteristic facies 1’ (IHPRF1), respectively. Although variants of uncertain significance (VUS) or presumed pathogenic variants have been studied in heterologous expression systems before, there is no generally accepted framework on how to assess or predict variant pathogenicity. We set out use the functional and computational characterization of 19 VUS detected in CLIFAHDD and IHPRF1 patients to establish a robust analysis to classify suspected disease-causing variants. Specifically, we employ a combination of multiple parameters derived from two-electrode voltage-clamp electrophysiology recordings and predicted protein stability and conservation scores. We show that this approach is capable of distinguishing benign common variants from both gain- and loss-of-function (GoF/LoF) variants. Additionally, our work provides mechanistic insight into the molecular mechanism underlying specific variants and provides insight into the unusual propensity of NALCN missense variants to result in GoF phenotypes. We anticipate that this experimental and computational framework will aid assessment of variant pathogenicity of NALCN and other components of the channelosome in the future.

The sodium leak channel, non selective (NALCN) is a member of the family of four-domain voltage-gated cation channels that include voltage-gated sodium (NaV) and calcium (CaV) channels [13, 23]. It possesses distinctive ion selectivity and pharmacological properties compared to these latter ion channels [6, 21]. NALCN, which is insensitive to tetrodotoxin (10 µM), has been proposed to mediate the tetrodotoxin-resistant and voltage-insensitive Na+ leak current (IL-Na) observed in many types of neurone [14]. However, whether NALCN is constitutively active has been challenged [20, 2, 7]. NALCN is widely distributed within the central nervous system and is also expressed in the heart and pancreas specifically, in rodents, within the islets of Langerhans [13, 14]. There is now strong functional and structural evidence indicating that NALCN forms a channelosome with obligatory auxiliary subunits UNC79, UNC80 and FAM155A (also known as NALF1) [6, 3, 12, 24, 10]. NALCN is the pore-forming α subunit, UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly that docks intracellularly onto NALCN. FAM155A forms an extracellular dome that shields extracellular access pathways to the selective filter of NALCN. There is also increasing evidence suggesting that the NALCN-UNC79-UNC80-FAM155A channelosome is modulated by additional auxiliary subunits including G proteins [18] and neuronal SNARE complex proteins [21]. However, there remain many areas of uncertainty surrounding NALCN function.It is worth noting that there is currently no NALCN-specific pharmacology. Inhibitors include multivalent cations (Gd3+, Ca2+, Mg2+, Ba2+, Zn2+) and small molecules (verapamil, 2-APB, DPBA, fluvastatin, L-703,606) [6, 11, 8, 19].

Proton-mediated activation of acid-sensing ion channel 1a (ASIC1a) leads to influx of sodium and, to a lesser extent, calcium, followed by rapid and complete desensitization. However, various ASIC1a-expressing cell types display atypical proton-induced currents characterized by altered proton sensitivity and slow and incomplete desensitization. The origin of this functional diversity and its physiological relevance remain unclear. Here, we show that the monocarboxylate transporter 1 (MCT1) interacts with ASIC1a, leading to subtype-specific modulation of proton sensitivity and desensitization. We delineate the molecular mechanism of the ASIC1a-MCT1 coupling and find that the presence of MCT1 is required for the distinct ASIC1a-mediated currents observed in glioblastoma cells, the most lethal brain cancer in adults. Strikingly, presence of ASIC1a together with MCT1 significantly prolongs the lifespan of mice intracranially injected with glioblastoma cells. We thus uncover the basis for altered ASIC1a function in glioblastoma and highlight the importance of deciphering the cell-type specific ASIC1a interactome.

NALCN (sodium leak channel, nonselective) is vital for regulating electrical activity in neurons and other excitable cells, and mutations in the channel or its auxiliary proteins lead to severe neurodevelopmental disorders. Here, we show that the neuronal SNARE (soluble N -ethylmaleimide–sensitive factor attachment protein receptors) complex proteins syntaxin and SNAP25 (synaptosome-associated protein 25), which enable synaptic transmission in the nervous system, inhibit the activity of the NALCN channel complex in both heterologous systems and primary neurons. The existence of this interaction suggests that the neurotransmitter release machinery can regulate electrical signaling directly and therefore modulate the threshold for its own activity. We further find that reduction of NALCN currents is sufficient to promote cell survival in syntaxin-depleted cells. This suggests that disinhibited NALCN may cause the puzzling phenomenon of rapid neuronal cell death in the absence of syntaxin. This interaction could offer opportunities for future drug development against genetic diseases linked to both NALCN- and SNARE protein–containing complexes.