Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation.

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.

Temporal changes in toe-brachial index results in haemodialysis patients.

INTRODUCTION: Toe brachial index (TBI), the ratio of toe pressure to systolic blood pressure (SBP), helps predict peripheral arterial disease. In patients with kidney failure this may be performed during haemodialysis for convenience. Until recently there has been little evaluation of the impact of haemodialysis in limb and systemic perfusion on these values. We aimed to determine if the values of TBI would change during and after dialysis compared to pre-dialysis assessments. METHODS: Using a repeated measures study, TBIs and toe pressures were measured using the Hadeco Smartop Vascular Ultrasound Doppler in 31 patients undergoing haemodialysis. TBI assessments were completed pre-dialysis and compared to values obtained at 1 hour, 2 hours, 3 hours, and post-dialysis to monitor change in TBI results. Comparison of values for each patient were tested for differences using paired t-tests. Linear mixed-effects models were used to test for the effect of patient and clinical factors on change in outcome measures. RESULTS: Mean TBI decreased from pre-dialysis at 1 hour (0.72 to 0.63, p = 0.01) and remained lower at 2 hours and 3 hours, before returning to pre-dialysis levels at post-dialysis. Mean systolic blood pressure also declined during dialysis. Mean TBI results were lower in those with a history of lower limb ulceration and in females. Sixteen patients (51.6%) had a normal TBI at baseline, 14 (45.2%) had a mildly low TBI, and one (3.2%) had a severely low TBI. Between baseline and 1 h, five patient's results moved from normal to mildly abnormal and one from mildly abnormal to severely abnormal. As haemodialysis concluded (post-dialysis) there were 17 (56.7%) 'normal' TBIs, with no severely abnormal TBIs (p = 0.73). 0.30). CONCLUSION: TBI and toe pressures are impacted significantly by dialysis. TBI and toe pressure assessments should be conducted before haemodialysis begins, or between dialysis sessions to avoid variability.

ATI-1777, a Topical Jak1/3 Inhibitor, May Benefit Atopic Dermatitis without Systemic Drug Exposure: Results from Preclinical Development and Phase 2a Randomized Control Study ATI-1777-AD-201.

Introduction: Atopic dermatitis, a chronic, pruritic skin disease, affects 10-30% of children and up to 14% of adults in developed countries. ATI-1777, a potent and selective Jak1/3 inhibitor, was designed with multiple sites of metabolism to deliver local efficacy in the skin and limit systemic exposure. In preclinical studies, ATI-1777 selectively inhibited Jak1/3 with limited systemic exposure and without any adverse effects. Primary objective: The primary goal of this study was to assess the preliminary clinical efficacy of ATI-1777 topical solution in adults with moderate or severe atopic dermatitis. Design: ATI-1777-AD-201, a phase 2a, first-in-human, randomized, double-blind, vehicle-controlled, parallel-group study, evaluated the efficacy, safety, tolerability, and pharmacokinetics of ATI-1777 topical solution in 48 participants with atopic dermatitis over 4 weeks. Primary endpoint: The primary endpoint was a reduction of a modified Eczema Area and Severity Index score from baseline. Results: Reduction was significantly greater in the ATI-1777-treated group on day 28 than in vehicle-treated group (percentage reduction from baseline = 74.45% [standard error = 6.455] and 41.43% [standard error = 6.189], respectively [P < .001]). Average plasma concentrations of ATI-1777 were <5% of the half-maximal inhibitory concentration of ATI-1777 for inhibiting Jak1/3. No deaths or serious adverse events were reported. Conclusion: Topical ATI-1777 does not lead to pharmacologically relevant systemic drug exposure and may reduce clinical signs of atopic dermatitis. Trial Registration: The study was registered at ClinicalTrials.gov with the number NCT04598269.

Electronic Polarizability Tunes the Function of the Human Bestrophin 1 Cl⁻ Channel.

Mechanisms of anion permeation within ion channels and nanopores remain poorly understood. Recent cryo-electron microscopy structures of the human bestrophin 1 chloride channel (hBest1) provide an opportunity to evaluate ion interactions predicted by molecular dynamics (MD) simulations against experimental observations. We implement the fully polarizable forcefield AMOEBA in MD simulations of open and partially-open states of the hBest1. The AMOEBA forcefield models multipole moments up to the quadrupole; therefore, it captures induced dipole and anion-π interactions. By including polarization we demonstrate the key role that aromatic residues play in ion permeation and the functional advantages of pore asymmetry within the highly conserved hydrophobic neck of the pore. We establish that these only arise when electronic polarization is included in the molecular models. We also show that Cl⁻ permeation in this region can be achieved through hydrophobic solvation concomitant with partial ion dehydration, which is compensated for by the formation of contacts with the edge of the phenylalanine ring. Furthermore, we demonstrate how polarizable simulations can help determine the identity of ion-like densities within high-resolution cryo-EM structures. Crucially, neglecting polarization in simulation of these systems results in the localization of Cl⁻ at positions that do not correspond with their experimentally resolved location. Overall, our results demonstrate the importance of including electronic polarization in realistic and physically accurate models of biological systems.

Transcending Markov: non-Markovian rate processes of thermosensitive TRP ion channels.

The Markov state model (MSM) is a popular theoretical tool for describing the hierarchy of time scales involved in the function of many proteins especially ion channel gating. An MSM is a particular case of the general non-Markovian model, where the rate of transition from one state to another does not depend on the history of state occupancy within the system, i.e. it only includes reversible, non-dissipative processes. However, an MSM requires knowledge of the precise conformational state of the protein and is not predictive when those details are not known. In the case of ion channels, this simple description fails in real (non-equilibrium) situations, for example when local temperature changes, or when energy losses occur during channel gating. Here, we show it is possible to use non-Markovian equations (i.e. offer a general description that includes the MSM as a particular case) to develop a relatively simple analytical model that describes the non-equilibrium behaviour of the temperature-sensitive transient receptor potential (TRP) ion channels, TRPV1 and TRPM8. This model accurately predicts asymmetrical opening and closing rates, infinite processes and the creation of new states, as well as the effect of temperature changes throughout the process. This approach therefore overcomes the limitations of the MSM and allows us to go beyond a mere phenomenological description of the dynamics of ion channel gating towards a better understanding of the physics underlying these processes.

Optical coherence tomography-based design for a real-time motion corrected scanning microscope.

While two-photon fluorescence microscopy is a powerful platform for the study of functional dynamics in living cells and tissues, the bulk motion inherent to these applications causes distortions. We have designed a motion tracking module based on spectral domain optical coherence tomography which compliments a laser scanning two-photon microscope with real-time corrective feedback. The module can be added to fluorescent imaging microscopes using a single dichroic and without additional contrast agents. We demonstrate that the system can track lateral displacements as large as 10 µm at 5 Hz with latency under 14 ms and propose a scheme to extend the system to 3D correction with the addition of a remote focusing module. We also propose several ways to improve the module's performance by reducing the feedback latency. We anticipate that this design can be adapted to other imaging modalities, enabling the study of samples subject to motion artifacts at higher resolution.

Correction: Limitations of non-polarizable force fields in describing anion binding poses in non-polar synthetic hosts.

Correction for 'Limitations of non-polarizable force fields in describing anion binding poses in non-polar synthetic hosts' by David Seiferth et al., Phys. Chem. Chem. Phys., 2023, 25, 17596-17608, https://doi.org/10.1039/D3CP00479A.

Limitations of non-polarizable force fields in describing anion binding poses in non-polar synthetic hosts.

Transmembrane anion transport by synthetic ionophores has received increasing interest not only because of its relevance for understanding endogenous anion transport, but also because of potential implications for therapeutic routes in disease states where chloride transport is impaired. Computational studies can shed light on the binding recognition process and can deepen our mechanistic understanding of them. However, the ability of molecular mechanics methods to properly capture solvation and binding properties of anions is known to be challenging. Consequently, polarizable models have been suggested to improve the accuracy of such calculations. In this study, we calculate binding free energies for different anions to the synthetic ionophore, biotin[6]uril hexamethyl ester in acetonitrile and to biotin[6]uril hexaacid in water by employing non-polarizable and polarizable force fields. Anion binding shows strong solvent dependency consistent with experimental studies. In water, the binding strengths are iodide > bromide > chloride, and reversed in acetonitrile. These trends are well captured by both classes of force fields. However, the free energy profiles obtained from potential of mean force calculations and preferred binding positions of anions depend on the treatment of electrostatics. Results from simulations using the AMOEBA force-field, which recapitulate the observed binding positions, suggest strong effects from multipoles dominate with a smaller contribution from polarization. The oxidation status of the macrocycle was also found to influence anion recognition in water. Overall, these results have implications for the understanding of anion host interactions not just in synthetic ionophores, but also in narrow cavities of biological ion channels.

Influence of electronic polarization on the binding of anions to a chloride-pumping rhodopsin.

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

Introduction to the shared near infrared spectroscopy format.

Significance: Functional near-infrared spectroscopy (fNIRS) is a popular neuroimaging technique with proliferating hardware platforms, analysis approaches, and software tools. There has not been a standardized file format for storing fNIRS data, which has hindered the sharing of data as well as the adoption and development of software tools. Aim: We endeavored to design a file format to facilitate the analysis and sharing of fNIRS data that is flexible enough to meet the community's needs and sufficiently defined to be implemented consistently across various hardware and software platforms. Approach: The shared NIRS format (SNIRF) specification was developed in consultation with the academic and commercial fNIRS community and the Society for functional Near Infrared Spectroscopy. Results: The SNIRF specification defines a format for fNIRS data acquired using continuous wave, frequency domain, time domain, and diffuse correlation spectroscopy devices. Conclusions: We present the SNIRF along with validation software and example datasets. Support for reading and writing SNIRF data has been implemented by major hardware and software platforms, and the format has found widespread use in the fNIRS community.

Gain-of-function mutations in KCNK3 cause a developmental disorder with sleep apnea.

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the 'X-gate', a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies.

When is a hydrophobic gate not a hydrophobic gate?

The flux of ions through a channel is most commonly regulated by changes that result in steric occlusion of its pore. However, ion permeation can also be prevented by formation of a desolvation barrier created by hydrophobic residues that line the pore. As a result of relatively minor structural changes, confined hydrophobic regions in channels may undergo transitions between wet and dry states to gate the pore closed without physical constriction of the permeation pathway. This concept is referred to as hydrophobic gating, and many examples of this process have been demonstrated. However, the term is also now being used in a much broader context that often deviates from its original meaning. In this Viewpoint, we explore the formal definition of a hydrophobic gate, discuss examples of this process compared with other gating mechanisms that simply exploit hydrophobic residues and/or lipids in steric closure of the pore, and describe the best practice for identification of a hydrophobic gate.

Structural basis for proton coupled cystine transport by cystinosin.

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

Influence of effective polarization on ion and water interactions within a biomimetic nanopore.

Interactions between ions and water at hydrophobic interfaces within ion channels and nanopores are suggested to play a key role in the movement of ions across biological membranes. Previous molecular-dynamics simulations have shown that anion affinity for aqueous/hydrophobic interfaces can be markedly influenced by including polarization effects through an electronic continuum correction. Here, we designed a model biomimetic nanopore to imitate the polar pore openings and hydrophobic gating regions found in pentameric ligand-gated ion channels. Molecular-dynamics simulations were then performed using both a non-polarizable force field and the electronic-continuum-correction method to investigate the behavior of water, Na+, and Cl- ions confined within the hydrophobic region of the nanopore. Number-density distributions revealed preferential Cl- adsorption to the hydrophobic pore walls, with this interfacial layer largely devoid of Na+. Free-energy profiles for Na+ and Cl- permeating the pore also display an energy-barrier reduction associated with the localization of Cl- to this hydrophobic interface, and the hydration-number profiles reflect a corresponding reduction in the first hydration shell of Cl-. Crucially, these ion effects were only observed through inclusion of effective polarization, which therefore suggests that polarizability may be essential for an accurate description for the behavior of ions and water within hydrophobic nanoscale pores, especially those that conduct Cl-.

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2.

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP2). Previous studies have demonstrated that PIP2 increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP2 remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP2-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP2 binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP2 to sites involving the proximal C-terminus, and we show that PIP2 promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP2 regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.

Water Nanoconfined in a Hydrophobic Pore: Molecular Dynamics Simulations of Transmembrane Protein 175 and the Influence of Water Models.

Water molecules within biological ion channels are in a nanoconfined environment and therefore exhibit behaviors which differ from that of bulk water. Here, we investigate the phenomenon of hydrophobic gating, the process by which a nanopore may spontaneously dewet to form a "vapor lock" if the pore is sufficiently hydrophobic and/or narrow. This occurs without steric occlusion of the pore. Using molecular dynamics simulations with both rigid fixed-charge and polarizable (AMOEBA) force fields, we investigate this wetting/dewetting behavior in the transmembrane protein 175 ion channel. We examine how a range of rigid fixed-charge and polarizable water models affect wetting/dewetting in both the wild-type structure and in mutants chosen to cover a range of nanopore radii and pore-lining hydrophobicities. Crucially, we find that the rigid fixed-charge water models lead to similar wetting/dewetting behaviors, but that the polarizable water model resulted in an increased wettability of the hydrophobic gating region of the pore. This has significant implications for molecular simulations of nanoconfined water, as it implies that polarizability may need to be included if we are to gain detailed mechanistic insights into wetting/dewetting processes. These findings are of importance for the design of functionalized biomimetic nanopores (e.g., sensing or desalination) as well as for furthering our understanding of the mechanistic processes underlying biological ion channel function.

Effects of ionic strength on gating and permeation of TREK-2 K2P channels.

In addition to the classical voltage-dependent behavior mediated by the voltage-sensing-domains (VSD) of ion channels, a growing number of voltage-dependent gating behaviors are being described in channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel, TREK2 responds to membrane voltage through a gating process mediated by the interaction of K+ with its selectivity filter. Recently, we found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found TREK-2 is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is complex.

Ion channels as convergence points in the pathology of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease of the cardiopulmonary system that lacks curative treatments. The main pathological event in PAH is elevated vascular resistance in the pulmonary circulation, caused by abnormal vasoconstriction and vascular remodelling. Ion channels are key determinants of vascular smooth muscle tone and homeostasis, and four PAH channelopathies (KCNK3, ABCC8, KCNA5, TRPC6) have been identified so far. However, the contribution of ion channels in other forms of PAH, which account for the majority of PAH patients, has been less well characterised. Here we reason that a variety of triggers of PAH (e.g. BMPR2 mutations, hypoxia, anorectic drugs) that impact channel function may contribute to the onset of the disease. We review the molecular mechanisms by which these 'extrinsic' factors converge on ion channels and provoke their dysregulation to promote the development of PAH. Ion channels of the pulmonary vasculature are therefore promising therapeutic targets because of the modulation they provide to both vasomotor tone and proliferation of arterial smooth muscle cells.

KCNK18 Biallelic Variants Associated with Intellectual Disability and Neurodevelopmental Disorders Alter TRESK Channel Activity.

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.

Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa.

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.