Spectator no more, the role of the membrane in regulating ion channel function

CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. Research funds for this study were also partly covered by a Tenovus Scotland grant (Tenovus Grant Application T15/41), awarded to CP. JHN is supported by the Chinese National Thousand Talents Program, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award.A pressure gradient across a curved lipid bilayer leads to a lateral force within the bilayer. Following ground breaking work on eukaryotic ion channels, it is now known that many proteins sense this change in the lateral tension and alter their functions in response. It has been proposed that responding to pressure differentials may be one of the oldest signaling mechanisms in biology. The most well characterized mechanosensing ion channels are the bacterial ones which open when the pressure differential hits a threshold. Recent studies on one of these channels, MscS, have developed a simple molecular model for how they sense and adapt to pressure. Biochemical and structural studies on mechanosensitive channels from eukaryotes have disclosed pressure sensing mechanisms. In this review, we highlight these findings and discuss the potential for a general model for pressure sensing.PostprintPeer reviewe

In-lipid structure of pressure sensitive domains hints mechanosensitive channel functional diversity

This project was supported by a BBSRC grant (BB/S018069/1) to C.P., who was supported by the Royal Society of Edinburgh, Tenovus (T15/41) and Carnegie Trust (OS000256), at the initial stages of this project. Further C.P. acknowledges support from the University of St Andrews for the C.K. studentship and the University of Leeds and the Chinese Scholarship Council for the Y.M. studentship. B.E.B. and C.P. acknowledge support by the Leverhulme Trust (RPG-2018–397). This work was also supported by previous Wellcome Trust [099149/Z/12/Z] and BBSRC equipment grants (BB/R013780/1).The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as structural model for rationalizing functional observations in multiple MscL orthologues. Although these orthologues adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nano-pockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here we modify the nano-pocket regions of MscL from Escherichia coli and Mycobacterium tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact EcMscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain EcMscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.Publisher PDFPeer reviewe

Sparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channels

BEB is grateful for funding from the European Union (Marie Curie Actions REA 334496). This work was supported by the EPSRC (EP/M024660/1) and the Wellcome Trust (099149/Z/12/Z). CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government.Pulse EPR is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g. homo-multimers) due to distorting multi-spin effects. Without suppression of these effects much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multi-spin problem.Here, two different appro aches for suppressing multi-spin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both, the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of E. coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multi-spin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analogue.Publisher PDFPeer reviewe

Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (T-m 0.7-1.5 degrees C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (T-m 5.0 +/- 1.7 degrees C and 3.0 +/- 1.8 degrees C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (T-m -4.7 +/- 1.1 degrees C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.Peer reviewe

HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

This project was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/S018069/1) to C.P., who also acknowledges support from the Wellcome Trust (WT) (219999/Z/19/Z) and the Chinese Scholarship Council (CSC) in the form of studentships for B.J.L. and B.W., respectively. A.N.C. is a Sir Henry Dale Fellow jointly funded by the WT and the Royal Society (220628/Z/20/Z). Funding from the BBSRC (BB/M012573/1) enabled the purchase of mass spectrometry equipment.Solvent accessibilities of and distances between protein residues measured by pulsed-EPR approaches provide high-resolution information on dynamic protein motions. We describe protocols for the purification and site-directed spin labeling of integral membrane proteins. In our protocol, peptide-level HDX-MS is used as a precursor to guide single-residue resolution ESEEM accessibility measurements and spin labeling strategies for EPR applications. Exploiting the pentameric MscL channel as a model, we discuss the use of cwEPR, DEER/PELDOR, and ESEEM spectroscopies to interrogate membrane protein dynamics. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).Publisher PDFPeer reviewe

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals

This work was supported by funds from the St Andrews EM facility and the University of St Andrews. CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. CP is also supported by a Tenovus Scotland Award (T15/41). JML was supported by a Programme grant from the Wellcome Trust (Number 045404).Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.Publisher PDFPeer reviewe

Monitoring the conformational ensemble and lipid environment of a mechanosensitive channel under cyclodextrin-induced membrane tension

This project was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/S018069/1) to C.P., who acknowledges support from the Wellcome Trust (WT) (219999/Z/19/Z) in the form of studentship for B.J.L. We also acknowledge support from the Chinese Scholarship Council (CSC) in the form of studentships for N.Y., Y.M., B.W., respectively. T.K.K. is supported by a Sir Henry Dale Fellowship funded by the WT and the Royal Society (223268/Z/21/Z). B.E.B. and C.P. thank the Leverhulme Trust (RPG-2018-397) for support. Funding from BBSRC (BB/R013780/1; BB/T017740/1) equipment grants enabled the purchase of the Qband Bruker pulse EPR spectrometer and University of Leeds funding the Bruker 950 MHz NMR spectrometer.Membrane forces shift the equilibria of mechanosensitive channels enabling them to convert mechanical cues into electrical signals. Molecular tools to stabilize and methods to capture their highly dynamic states are lacking. Cyclodextrins can mimic tension through the sequestering of lipids from membranes. Here we probe the conformational ensemble of MscS by EPR spectroscopy, the lipid environment with NMR, and function with electrophysiology under cyclodextrin-induced tension. We show the extent of MscS activation depends on the cyclodextrin-to-lipid ratio, and that lipids are depleted slower when MscS is present. This has implications in MscS’ activation kinetics when distinct membrane scaffolds such as nanodiscs or liposomes are used. We find MscS transits from closed to sub-conducting state(s) before it desensitizes, due to the lack of lipid availability in its vicinity required for closure. Our approach allows for monitoring tension-sensitive states in membrane proteins and screening molecules capable of inducing molecular tension in bilayers.Peer reviewe

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a molecular scissor that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex

Using biophysical techniques to study the mechanism of ligand-gated potassium efflux systems (KEF) from bacteria

The ligand-gated potassium channels KefC and KefB of Escherichia coli are critical components in protecting cells from toxic electrophiles.  Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles.  KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis.  In vivo K+ measurements were performed to ensure that all of the constructs used were fully functional.  Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements in vivo, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data.  In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating.  Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC.  Finally, a variety of chemically diverse glutathione adducts was tested on KefC in vitro to elucidate the mechanism by which these ligands initiate K+ flux through the associated transmembrane domain.EThOS - Electronic Theses Online ServiceGBUnited Kingdo