Biology subjects

Pless, S.

Publications and source records attributed to Pless, S..

7 recordsLinked to original sources

bioRxiv2026

A deep learning-generated hASIC1a miniprotein inhibitor confers neuroprotection in stroke

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.

biophysics↗

bioRxiv2026

Protein semi-synthesis enables real-time optical tracking of intracellular conformational changes during sodium channel inactivation

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.

biophysics↗

bioRxiv2026

Preferential vulnerability of cortical GABAergic interneurons to Nalcn deficiency

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.

neuroscience↗

bioRxiv2025

Framework for combined functional and computational assessment of variant pathogenicity in the sodium leak channel NALCN

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.

genetics↗

bioRxiv2023

Unplugging DI-DIV and DII-DIII lateral fenestrations of NALCN reveals unexpected pharmacology

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that oc-cluded lateral fenestrations underlie the pharmacological resistance of NALCN to lipophilic compounds, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned resi-dues with alanine (AAAA) and compared the effects of NaV, CaV and NALCN block-ers on both wild-type (WT) and AAAA channels. Most compounds behaved in a simi-lar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cav-ity through distinct fenestrations, implying structural specificity to their modes of ac-cess. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug bind-ing site(s) within the pore region. Intrigued by the activity of 2-APB and its ana-logues, we tested additional compounds containing the diphenylmethane/amine moiety on WT channels. We identified compounds from existing clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. Significance statementThe sodium leak channel (NALCN) is essential for survival: mutations cause life-threatening developmental disorders in humans. However, no treatment is currently available due to the resistance of NALCN to pharmacological targeting. One likely reason is that the lateral fenestrations, a common route for clinically used drugs to enter and block related ion channels, are occluded in NALCN. Using a combination of computational and functional approaches, we unplugged the fenestrations of NALCN which led us to the first molecularly defined drug binding site within the pore region. Besides that, we also identified additional NALCN modulators from existing clinically used therapeutics, thus expanding the pharmacological toolbox for this leak channel.

pharmacology and toxicology↗

bioRxiv2020

An unconventional conductance is required for pacemaking of nigral dopamine neurons

Although several ionic mechanisms are known to control rate and regularity of the pacemaker in dopamine (DA) neurons from the substantia nigra pars compacta (SNc), a conductance essential for pacing has yet to be defined. Here we provide pharmacological evidence that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidine (XG), an established blocker of gating pore currents in mutant voltage gated sodium channels, selectively stops pacemaking of DA SNc neurons and is without effect on the main pore of their voltage-gated channels. We isolated a voltage-dependent, non-inactivating XG-sensitive current of 20-25 pA which operates in the relevant subthreshold range and is carried by both Na+ and Cl- ions. While the molecular identity of this conductance remains to be determined, we show that this XG-sensitive current is crucial to sustain pacemaking in these neurons.

neuroscience↗

bioRxiv2019

Mechanism and site of action of big dynorphin on ASIC1a

Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics. Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

biophysics↗