Aug 2026· The Journal of General Physiology· Vol 158· 0 citations
Medicine
TL;DR
A stratified molecular model of TMEM63B channelopathies in which the pathogenic variants progressively destabilize the hydrophobic gate, permitting lipid permeation at the resting state followed by force-induced ion and lipid co-transport through I475del is supported.
Abstract
TMEM63B, a mechanosensitive ion channel (MSC), is associated with severe neurodevelopmental disorders, including severe early-onset developmental and epileptic encephalopathy (DEE). Structure-function studies have shown that TMEM63B pathogenic variants, including V44M in transmembrane helix 0 (TM0) and T481N in TM4, cluster near the hydrophobic neck region of the ion permeation pathway, a region critical for gating and permeation. Notably, V44M and T481N convert TMEM63B into constitutive phospholipid scramblases without obvious effects on their MSC activity, revealing an unexpected channel-to-scramblase switch in these variants. To further define the mechanistic basis of this phenomenon, here we characterized I475del, a TM4 deletion variant near V44M and T481N. Unlike V44M and T481N, the I475del channel exhibited basal leak currents, enhanced mechanically activated currents, and elevated mechanosensitivity, altogether supporting its classification as a bona fide gain-of-function MSC variant. Like V44M and T481N, I475del also enabled constitutive phospholipid scramblase activity. However, unlike these variants, I475del uniquely displayed further potentiation of scramblase activity under hypotonic osmotic stress. In addition, mutating the key residues that control gating conformational changes abolished gain-of-function ion and lipid transport through I475del. Together, our results support a stratified molecular model of TMEM63B channelopathies in which the pathogenic variants progressively destabilize the hydrophobic gate, permitting lipid permeation at the resting state followed by force-induced ion and lipid co-transport. These findings advance the mechanistic understanding of TMEM63B function and TMEM63B-associated disease and provide a framework for developing therapeutic strategies targeting variant-specific pathologies.
The mechanosensitive channel of small conductance (MscS) is the founding member of the family of MscS-like channels, which share a structurally conserved core but feature additional structural elements that define their specific channel characteristics. Here, we characterize the structure and function of the Escherichia coli mechanosensitive channel of mini conductance (MscM), which features eight additional transmembrane (TM) helices and a large periplasmic domain. Our cryo-EM structures reveal that channel gating involves conformational changes in all domains of MscM. In particular, a cytoplasmic extension of TM7 couples the conformation of the TM domain to that of the cytoplasmic domain, resulting in gating of its lateral fenestrations, where ions enter the channel. Thus, different from all other MscS-like channels studied to date, channel gating in MscM is mediated by its cytoplasmic domain and not the TM domain, which senses changes in membrane tension and operates the cytoplasmic gates. Mechanosensitive channels in bacteria provide protection against hypoosmotic shock. Here, the authors use cryo-electron microscopy to reveal the gating mechanism of MscS-like channel EcMscM, showing that the conformations of the transmembrane and cytoplasmic domains are coupled.
Giorgos Hiotis, T. Walz· Nature Communications· 0 citations
Mechanosensitive channels of large conductance (MscL) protect bacteria from hypo-osmotic stress by opening a large pore in response to membrane tension. The transmembrane TM1 and TM2 helices together with the N-terminal amphipathic helix function as the primary force-sensing structural domains, whereas the role of the periplasmic loop in the channel gating remains poorly understood. Using experimental and computational approaches, our study demonstrates that mutating the loop residues (A64, Q65, G66, D67) or inserting a four-glycine flexible hinge at D67 site modified the properties of the channel recorded in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids. Extending the periplasmic loop disrupted mechanical force transmission, reducing channel sensitivity to applied tension. Q65R channel mutant exhibited increased sensitivity in azolectin liposomes that was reduced in negatively charged liposomes and spheroplast membranes. The Q65E mutant showed decreased sensitivity across all preparations tested and exhibited channel flickering in negatively charged liposomes. Molecular dynamics simulations revealed that Q65E produced larger but more structurally restricted conformational changes, whereas wild-type MscL and Q65R exhibited rapid and extensive pore opening over a shorter timeframe. Our findings establish the periplasmic loop as a structural domain fine-tuning MscL gating through electrostatic interactions with surrounding lipids.
K. Duru, Paul Rohde, H. Hafezi et al.· npj Biological Physics and M...· 0 citations
Endolysosomal abnormalities are particularly detrimental to the nervous system and have been implicated in neuropsychiatric disorders. Key regulators of the lysosomal and endosomal luminal ion homeostasis are CLC chloride/proton exchangers. We report 15 individuals carrying variants in CLCN3, encoding a ubiquitous endosomal 2Cl−/H+ exchanger, and provide updated clinical information for 5 previously reported individuals. Subjects displayed a broad spectrum of neuropsychiatric symptoms, including developmental delay, intellectual disability, and epilepsy. To reveal the pathogenic mechanism, we investigated ClC-3 variants-mediated ion transport and its regulation by the recently discovered inhibitory beta subunit TMEM9. 12/20 missense variants exhibited altered properties and fell into two classes: those affecting the region binding inhibitory TMEM9 carboxy-termini, and those that broaden the voltage range over which ClC-3 conducts ions. Surprisingly, the latter variants also attenuated TMEM9-mediated inhibition. Both classes produced a toxic gain-of-function, as evident from endolysosomal vacuolization by mutant ClC-3/TMEM9 overexpression. Our results expand the genetic and clinical spectrum of CLCN3-related disease, provide a solid basis for genetic counseling, and uncover an unexpected link between gating-associated conformational changes and inhibition by TMEM9. Loss- and gain-of-function variants of the endosomal chloride/proton exchanger ClC-3 are associated with neurodevelopmental disorders. Identification and characterization of novel variants expands the clinical spectrum of CLCN3 disease and provides detailed insights into pathogenic mechanisms. Most heterozygous missense variants result in a gain of function when studied in co-expression with TMEM9 β-subunits. Several variants affect ClC-3 residues close to the binding pocket of the TMEM9 carboxy-terminus that directly blocks the chloride pathway, thereby weakening the block. Several other variants, located far from the binding site, affect voltage-dependent gating when studied without TMEM9, thereby enhancing currents at endosomal voltages. These variants also weaken TMEM9-mediated inhibition, revealing a link between gating-associated conformational changes and TMEM9 binding. Most heterozygous missense variants result in a gain of function when studied in co-expression with TMEM9 β-subunits. Several variants affect ClC-3 residues close to the binding pocket of the TMEM9 carboxy-terminus that directly blocks the chloride pathway, thereby weakening the block. Several other variants, located far from the binding site, affect voltage-dependent gating when studied without TMEM9, thereby enhancing currents at endosomal voltages. These variants also weaken TMEM9-mediated inhibition, revealing a link between gating-associated conformational changes and TMEM9 binding. Loss- and gain-of-function variants of the endosomal chloride/proton exchanger ClC-3 are associated with neurodevelopmental disorders. Identification and characterization of novel variants expands the clinical spectrum of CLCN3 disease and provides detailed insights into pathogenic mechanisms.
Maya M. Polovitskaya, T. Tkemaladze, L. Jensen et al.· EMBO Molecular Medicine· 0 citations
ABSTRACT Plasma membrane lipid asymmetry is tightly regulated and fundamental to mammalian cell physiology. TMEM30A is the β-subunit of P4-ATPases, flippase enzymes that maintain strict phosphatidylserine (PS) asymmetry by pumping it from the outer to the cytosolic leaflet. Loss of TMEM30A function causes constitutive PS externalization and has been implicated in diseases such as diffuse large B-cell lymphoma and tumor immune evasion. Here, we systematically define the biophysical and molecular consequences of TMEM30A deletion in immune cells. Using a live-cell lipid reporter, membrane order probe, and surface proteome mapping, we show that TMEM30A-knockout cells display robust PS externalization accompanied by faster lateral diffusion of membrane constituents and decreased plasma membrane order. Surface proteome reorganization includes increased abundance of tetraspanins and CD47. Furthermore, TMEM30A loss triggers glycocalyx remodeling via ADAM10-dependent shedding, which removes major transmembrane mucins, including CD43 and CD162 (also known as SPN and SELPLG, respectively). Together, these data reveal a coordinated reorganization of lipids, glycans and proteins upon TMEM30A loss, suggesting mechanistic links between flippase dysfunction and increased plasma membrane dynamics and potential sensitization to immune therapy. Furthermore, our study provides an integrated surfaceome framework that might shed light on the relationship between TMEM30A expression and clinical outcomes in cancer.
C. Gurdap, F. Ragaller, Marion Muller et al.· Journal of Cell Science· 0 citations
A generic numbering scheme for TMEM16 scramblases (GNS-TMEM16), modeled on the Ballesteros & Weinstein system established for class A G protein-coupled receptors, is introduced, bringing to light the advantages of corresponding residues identification in different TMEM16 proteins and show that the mammalian scramblase undergoes substantially larger separation at the extracellular groove entrance than either fungal homolog.
The interaction between mammalian enabled protein (Mena), an actin regulatory protein, and protein tyrosine phosphatase 1B (PTP1B) is critical for maintaining epidermal growth factor receptor (EGFR) signaling homeostasis and regulating cellular motility. Mena recruits PTP1B to activated EGFR in vivo, facilitating receptor dephosphorylation and limiting invasive signaling. However, overexpression of wild-type Mena or the invasive isoform, MenaINV, perturbs this regulatory mechanism by altering PTP1B localization and EGFR signaling dynamics. Here, we report the solution NMR structure of the unliganded Mena EVH1 domain, which contains a conserved aromatic triad (Y16, W23, F77) that forms the canonical polyproline-binding cleft. Building on prior evidence that the PTP1B polyproline region serves as an EVH1 ligand, we investigated the structural basis of the interaction between EVH1 and wild-type PTP1B. NMR chemical shift perturbation analyses using multiple PTP1B variants, including full-length and truncated constructs, revealed that binding affects not only the canonical EVH1 hydrophobic cleft but also residues on the opposite surface, suggesting an expanded interaction interface. Diffusion-ordered spectroscopy (DOSY) and size-exclusion chromatography further indicate that PTP1B variants containing the disordered C-terminal tail form more compact complexes with EVH1, consistent with a disorder-to-order transition. Complementary perturbation studies using [2H,15N] PTP1B demonstrate that EVH1 binding induces changes not only in the polyproline region but also across the phosphatase core, α7 helix, and C-terminal tail. Despite these structural perturbations, enzymatic assays show that EVH1 binding does not affect the catalytic activity of PTP1B, supporting a model in which Mena functions as a scaffold to spatially organize PTP1B within EGFR signaling complexes.
Lanette LaComb, Sean M. Cahill, J. Bonanno et al.· Biochemistry· 0 citations
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