Olivier Bignucolo

Inherited mutations in the KCNH2 gene, which encodes the cardiac hERG potassium channel, are major contributors to arrhythmogenic syndromes such as long QT and short QT syndromes. However, clinical interpretation of the growing number of missense variants – many of which are classified as variants of uncertain significance (VUS) – remains a pressing challenge. Here, we present a semi-automated in silico pipeline for predicting hERG variant pathogenicity, acting as a binary classifier and integrating five structural metrics – residue volume, hydrophobicity, charge, steric clashes, and proximity to pathogenic hotspots – into a composite structural pathogenicity score (SPS) scaled from 1 to 5. Applied to 1727 hERG variants from ClinVar and from a French nationwide cohort, this binary classifier, termed SPARC, identified 260 variants as high risk of pathogenicity with SPS ≥3.25, of which a representative subset from the French cohort was functionally validated using high-throughput automated patch-clamp. Functional phenotyping confirmed the structural predictions, including for several VUS, demonstrating that comprehensive structural scoring can reliably stratify variant pathogenicity. This approach, benchmarked with Alpha Missense and Revel, offers a superior scalable, cost-effective pre-screening tool to guide clinical variant interpretation and prioritization for experimental validation.

Acid-sensing ion channels (ASICs) are neuronal Na ⁺ -permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca ²⁺ concentration decreases the H ⁺ sensitivity of ASIC1a, suggesting a competition for binding sites between H ⁺ and Ca ²⁺ ions. Here, we predicted candidate residues for Ca ²⁺ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca ²⁺ of the ASIC1a pH dependence of activation and desensitization. This occurred probably owing to a disruption of Ca ²⁺ binding. Our results link one of the two predicted Ca ²⁺ -binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg ²⁺ regulates ASICs in a similar way as does Ca ²⁺ . We show that Mg ²⁺ shares some of the binding sites with Ca ²⁺ . Finally, we provide evidence that some of the ASIC1a Ca ²⁺ -binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca ²⁺ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

Highly selective for K⁺ at neutral pH, the TWIK1 channel becomes permeable to Na⁺ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5–6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K⁺-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na⁺-permeable conformation of the channel.

Acid-sensing ion channels (ASICs) are neuronal H ⁺ -gated, Na ⁺ -permeable channels involved in learning, fear sensing, pain sensation and neurodegeneration. An increase in the extracellular Ca ²⁺ concentration shifts the pH dependence of ASIC1a to more acidic values. Here, we predicted candidate residues for Ca ²⁺ binding on ASIC1a, based on available structural information and molecular dynamics simulations; the function of channels carrying mutations of these residues was then measured. We identify several residues in cavities previously associated with pH-dependent gating, whose mutation decreased the Ca ²⁺ -induced shift in ASIC1a pH dependence, likely due to a disruption of Ca ²⁺ binding. We show also that Mg ²⁺ shares some of the binding sites with Ca ²⁺ , and that some of the Ca ²⁺ binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation binding sites in ASIC1a shows how Ca ²⁺ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

At neutral pH, the TWIK1 channel is highly selective for K ⁺ . When exposed to acidification, it becomes permeable to Na ⁺ . This change occurs within minutes and is reversible. By combining pKa calculations, molecular dynamics (MD) simulations, mutagenesis and electrophysiology, we identified a network of residues involved in this unique property. MD simulations captured crucial features associated with channel gating and previously observed by cryogenic electron microscopy (cryo-EM) at pH7.4 and pH5, such as the pH-dependent orientation of the lateral side chain of the proton sensor His122 and the elongation of the entire pore structure upon acidification. Between the closed and open states of TWIK1 observed by cryo-EM, MD simulations show that the channel undergoes additional conformational changes between pH 7.5 to 6 that involves the His122, Glu235, Lys246 and Phe109 residues. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K ⁺ -selective state at high pH, disappear upon acidification. This causes dissociation of the Phe109 aromatic side chain from this network, ultimately leading to the Na ⁺ -permeable conformation of the channel.

The extracellular Ca ²⁺ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca ²⁺ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H ⁺ -gated Na ⁺ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca ²⁺ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca ²⁺ . Two previous studies concluded that low pH opens ASIC3 by displacing Ca ²⁺ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca ²⁺ . Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca ²⁺ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

A central problem in computational biophysics is the treatment of titratable residues in molecular dynamics simulations of large biological macromolecular systems. Conventional simulation methods ascribe a fixed ionization state to titratable residues in accordance with their pKa and the pH of the system, assuming that an effective average model will be able to capture the predominant behavior of the system. While this assumption may be justifiable in many cases, it is certainly limited, and it is important to design alternative methodologies allowing a more realistic treatment. Constant-pH simulation methods provide powerful approaches to handle titratable residues more realistically by allowing the ionization state to vary statistically during the simulation. Extending the molecular mechanical (MM) potential energy function to a family of potential functions accounting for different ionization states, constant-pH simulations are designed to sample all accessible configurations and ionization states, properly weighted according to their Boltzmann factor. Because protonation and deprotonation events correspond to a change in the total charge, difficulties arise when the long-range Coulomb interaction is treated on the basis of an idealized infinite simulation model and periodic boundary conditions with particle-mesh Ewald lattice sums. Charging free-energy calculations performed under these conditions in aqueous solution depend on the Galvani potential of the bulk water phase. This has important implications for the equilibrium and nonequilibrium constant-pH simulation methods grounded in the relative free-energy difference corresponding to the protonated and unprotonated residues. Here, the effect of the Galvani potential is clarified, and a simple practical solution is introduced to address this issue in constant-pH simulations of the acid-sensing ion channel (ASIC).

Acid-sensing ion channels (ASICs) are neuronal Na⁺ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation, and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here, we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

Acid-sensing ion channels (ASICs) are neuronal Na ⁺ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. Molecular dynamics simulations were used to further evaluate VCF-predicted movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

Voltage-gated potassium channels (Kv) allow ion permeation upon changes of the membrane electrostatic potential (Vm). Each subunit of these tetrameric channels is composed of six transmembrane helices, of which the anti-parallel helix bundle S1-S4 constitutes the voltage-sensor domain (VSD) and S5-S6 forms the pore domain. Here, using 82 molecular dynamics (MD) simulations involving 266 replicated VSDs, we report novel responses of the archaebacterial potassium channel KvAP to membrane polarization. We show that the S4 α-helix, which is straight in the experimental crystal structure solved under depolarized conditions (Vm ∼ 0), breaks into two segments when the cell membrane is hyperpolarized (Vm << 0), and reversibly forms a single straight helix following depolarization (Vm = 0). The outermost segment of S4 translates along the normal to the membrane, bringing new perspective to previously paradoxical accessibility experiments that were initially thought to imply the displacement of the whole VSD across the membrane. The novel model is applied through steered and unbiased MD simulations to the recently solved whole structure of KvAP. The simulations show that the resting state involves a re-orientation of the S5 α-helix by ∼ 5–6 degrees in respect to the normal of the bilayer, which could result in the constriction and closure of the selectivity filter. Our findings support the idea that the breakage of S4 under (hyper)polarization is a general feature of Kv channels with a non-swapped topology.