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C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918) The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment. Publisher PDF

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 PDF

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity., How mechanosensitive ion channels, such as MscL, are activated by lipids and physical properties of the membrane remains unclear. Here authors use PELDOR/DEER spectroscopy and identify a single site which generated an allosteric structural response in the absence of membrane tension.

The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.

International audience; 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.

Pulsed EPR methods, such as PELDOR (or DEER) and ESEEM have emerged as powerful tools for assessing membrane protein conformation, folding, oligomerisation and dynamics. PELDOR distance measurements can provide quantitative information on the conformational equilibria of membrane proteins, dovetailing well with techniques such as X-ray crystallography and cryo-EM. ESEEM spectroscopy can offer deuterium (or solvent) accessibility quantification at a single residue level. An inherent limit placed on membrane protein studies is the need to remove the protein from its natural membrane environment. However, recent developments in the membrane protein field provide lipid scaffolds that mimic that environment, to limit the impact of protein solubilisation and offer flexibility of choice in lipid composition, a key factor in the functional integrity of membrane proteins. Additionally, computational tools can be used to aid spin-labelling experiments, or tailored to incorporate experimentally derived PELDOR distance restraints to reveal the complete conformational ensemble of membrane proteins. In this chapter, we will focus on the application of pulsed EPR in the study of membrane proteins, highlighting case studies including our work on mechanosensitive ion channels. We will discuss experimental considerations, and introduce some of the computational tools that can be used in combination with pulsed EPR methods.

Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium

Funding: CP would like to acknowledge the Royal Society of Edinburgh, Tenovus (T15/41), Carnegie Trust (OS000256) grant awards and the School of Biology of the University of St Andrews for a PhD studentship to CK. Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium Escherichia coli has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane protein production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields. In previous work, we generated the engineered E. coli strains SuptoxD and SuptoxR, which upon coexpression of the effector genes djlA or rraA, respectively, can suppress the cytotoxicity caused by MP overexpression and produce enhanced MP yields. Here, we systematically looked for gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains. We have found that, under optimal conditions, SuptoxD and SuptoxR achieve greatly enhanced recombinant production for a variety of MP, irrespective of their archaeal, eubacterial, or eukaryotic origin. Furthermore, we demonstrate that the use of these engineered strains enables the production of well-folded recombinant MPs of high quality and at high yields, which are suitable for functional and structural studies. We anticipate that SuptoxD and SuptoxR will become broadly utilized expression hosts for recombinant MP production in bacteria. Postprint

This work was supported by Wellcome Trust grants WT092552MA (J.H.N. and I.R.B.), Senior Investigator Award WT100209MA (J.H.N.), 093228 (T.K.S.) and 092970 (M.S.P.S.), and Biotechnology and Biological Sciences Research Council grants BB/I019855/1 (M.S.P.S.), BB/H017917/1 (J.H.N. and I.R.B.) and BB/J009784/1 (H.B.). I.R.B. is supported as a Leverhulme Emeritus Fellow. J.H.N. is supported as a Royal Society Wolfson Merit Award holder and as a 1000 Talent Scholar at Sichuan University. A.C.E.D. was supported by an Engineering and Physical Sciences Research Council Systems Biology Doctoral Training Centre student fellowship. The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates. Postprint Postprint Postprint Postprint Postprint Postprint Postprint

This work was supported by the University of St Andrews and Nanomorphomics group funds. The work forms part of an International Patent Application No. PCT/GB2015/052482. Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM. Publisher PDF

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail.

Highlights • Uranyl acetate/tungsten double stains are proposed for imaging lipid rich nanoparticle in TEM. • Combined with methylcellulose embedment, the technique enhances membrane contrast. • The technique works for liposomes, nanodiscs and bicelles. • The double staining should improve quantification of lipid rich nanoparticles., 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.

This work was supported by The Wellcome Trust (WT092552MA), The Leverhulme Trust (EM-2012-60\2), BBSRC SysMO (BB/F003455/1), a European Union Marie Curie ITN Award (NICHE; 289384) The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of 10–20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10–20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system. Publisher PDF

Funding: EaStCHEM studentship to E.B. The work was funded by BBSRC grant BB/H017917/1 to JNH, OS & IRB, The Leverhulme Foundation (EM-2012-60\2) and equipment from a Wellcome Trust Capital Award. Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore- forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating. Publisher PDF

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail.

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.

Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses

Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.

The heptameric mechanosensitive channel of small conductance (MscS) provides an essential function in Escherichia coli as it prevents cell lysis by opening in response to increased bilayer tension, during extreme osmotic shock. The gating of MscS has been extensively studied by three approaches: X-ray crystallography [1], cwEPR [2] spectroscopy and EMD simulations [3], but each of them has defined different closed and open structures of the channel. The proposed models report very different arrangements of the transmembrane helices. To resolve the discrepancy, we have attached spin labels to cysteine mutants on key structural elements within all three MscS transmembrane helices, specifically chosen to discriminate between the competing models. These distances from multiple mutants provide an independent assessment of the validity of the competing models. In addition, they offer the advantage of measurements on MscS in solution under physiological conditions. The resulting pulsed electron-electron double resonance (PELDOR) spectra allow the accurate measurement of transmembrane helix separation, offering a direct comparison for the different models. The distance distributions for the open crystal structure of MscS match the experimental data [4]. We also report two new crystal structures of spin labeled MscS which are in complete agreement with the PELDOR data and demonstrate the accuracy of PELDOR for complex ion channels. PELDOR is a powerful experimental tool which can be used for interrogating the conformation of transmembrane regions of multimeric membrane proteins.[1] Wang et al., Science 321, 1179 (2008).[2] Vasquez et al., Science 321, 1210 (2008).[3] Akitake et al., NSMB 14, 1141 (2007).[4] Pliotas et al., PNAS (doi/10.1073/pnas.1202286109) (2012)

Electrophiles react rapidly with DNA and proteins and are therefore toxic. Bacteria protect themselves from the immediate effects of electrophiles by lowering the cytosolic pH before the compounds are detoxified. This is achieved by the Kef systems, which mediate potassium efflux, in turn coupled to an influx of protons. We investigated the activation and complex regulation of this system. When electrophiles enter the cell, the most likely reaction partner is the nucleophile glutathione which is present at high concentration in the cytosol. The resulting glutathione adducts activate Kef while glutathione itself inhibits. The kinetics of activation was investigated in vivo using a potassium selective electrode. Activator and inhibitor bind to the same binding site on the regulatory KTN domain of Kef and cause differential structural changes, as shown by crystal structures of this domain [1]. We characterised the binding and these structural changes further using fluorescence and EPR spectroscopy in purified Kef proteins. A second binding site on the KTN domain binds nucleotides. Furthermore, Kef is regulated by a soluble protein which serves as activator [2]. We present data that this activator protein also has quinone oxidoreductase activity. The relationship between this enzymatic activity and its function as an activator of Kef was dissected using point mutations to disrupt enzyme activity but preserve structural integrity.[1] Rossild et al., Structure 17, 893 (2009).[2] Miller et al., J. Bac.182, 6536 (2000).

Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied. MscS-like channels have been found in plants and Schizosaccharomyces pombe, where they regulate intracellular Ca2+ and cell volume under hypo-osmotic conditions. Here we characterize two MscS-like putative channels, named MscA and MscB, from the model filamentous fungus Aspergillus nidulans. Orthologues of MscA and MscB are present in most fungi, including relative plant and animal pathogens. MscA/MscB and other fungal MscS-like proteins share the three transmembrane helices and the extended C-terminal cytosolic domain that form the structural fingerprint of MscS-like channels with at least three additional transmembrane segments than Escherichia coli MscS. We show that MscA and MscB localize in Endoplasmic Reticulum and the Plasma Membrane, respectively, whereas their overexpression leads to increased CaCl2 toxicity or/and reduction of asexual spore formation. Our findings contribute to understanding the role of MscS-like channels in filamentous fungi and relative pathogens.