Journal of General Physiology最新文献_第2页

Dissecting diastolic stiffness. 解剖舒张刚度。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-11-02 Epub Date: 2026-10-19 DOI: 10.1085/jgp.202614130

Ben Short

引用次数: 0

A fluorescent non-hydrolyzable probe for the nucleotide-binding sites of KATP. 一种用于检测KATP核苷酸结合位点的荧光非水解探针。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-11-02 Epub Date: 2026-09-18 DOI: 10.1085/jgp.202513840

Priscilla Rubio, Shayla Q Whitaker, Jonathan Ashby, Michael C Puljung

{"title":"A fluorescent non-hydrolyzable probe for the nucleotide-binding sites of KATP.","authors":"Priscilla Rubio, Shayla Q Whitaker, Jonathan Ashby, Michael C Puljung","doi":"10.1085/jgp.202513840","DOIUrl":"10.1085/jgp.202513840","url":null,"abstract":"Neuroendocrine ATP-sensitive K+ channels (KATP) comprise four pore-forming subunits (Kir6.2), each associated with a modulatory sulfonylurea receptor subunit (SUR1). ATP/ADP binding to Kir6.2 inhibits KATP; MgATP/MgADP binding to two different sites on SUR1 promotes activation. As SUR1 is a member of the ABC transporter family of proteins, it can potentially hydrolyze MgATP to MgADP. Whether this activity is required for KATP activation remains controversial. Previous studies demonstrated that non-hydrolyzable ATP analogs do not activate KATP, which may reflect an inability of these compounds to bind to SUR1, their inability to promote a conformational change in SUR1 that leads to channel activation, or a requirement for ATP hydrolysis during channel gating. To explore this further, we synthesized a fluorescent trinitrophenyl (TNP) derivative of the non-hydrolyzable ATP analog β,γ-methyleneadenosine 5'-triphosphate (AMP-PCP). Synthesis was verified by UV-visible absorbance, fluorescence spectroscopy, 1H NMR, and mass spectrometry. Purity was assessed by reversed-phase HPLC. Real-time nucleotide binding to intact KATP channels in cell membranes was measured using FRET between channels labeled with a fluorescent, noncanonical amino acid and TNP-nucleotide derivatives. This technique provides sufficient spatial resolution to discriminate between binding to each site on KATP. Using this approach, we first established that TNP-ATP can bind to nucleotide-binding site (NBS) 1 on SUR1 in fluorescently labeled Kir6.2/SUR1 channels in unroofed membranes of HEK293T cells. We subsequently demonstrated that TNP-AMP-PCP binds to both NBSs on SUR1 in the absence of Mg2+. AMP-PCP was able to compete with TNP-ATP for binding to NBS2, suggesting that it, too, binds NBS2. We conclude that the failure of non-hydrolyzable ATP analogs to activate KATP does not stem from an inability of these nucleotides to bind to the channel.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13629173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148992312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The many faces of Orai1 dysfunction: Molecular mechanisms and clinical manifestations. Orai1功能障碍的多个方面：分子机制和临床表现。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-11-02 Epub Date: 2026-08-31 DOI: 10.1085/jgp.202614031

Magdalena Prantl, Lara Atzgerstorfer, Tamara Radiskovic, Marc Fahrner, Isabella Derler

{"title":"The many faces of Orai1 dysfunction: Molecular mechanisms and clinical manifestations.","authors":"Magdalena Prantl, Lara Atzgerstorfer, Tamara Radiskovic, Marc Fahrner, Isabella Derler","doi":"10.1085/jgp.202614031","DOIUrl":"https://doi.org/10.1085/jgp.202614031","url":null,"abstract":"Orai1, the pore-forming subunit of the calcium (Ca2+) release-activated Ca2+ (CRAC) channel, plays a central role in store-operated Ca2+ entry (SOCE) in animal cells and thereby serves as a key regulator of intracellular Ca2+ homeostasis. Disruption of this tightly controlled process is associated with a wide spectrum of human diseases. Dysfunction can result from a multitude of remodeling mechanisms, including altered protein expression (up- or downregulation), assembly remodeling, or mutations. We focus in particular on Orai1 mutations, which have been linked to severe combined immunodeficiency (SCID) as a result of channel loss-of-function (LoF), as well as to disorders like tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) arising from gain-of-function (GoF) alterations. These mutation-induced functional defects can be attributed to a wide variety of disruptions in the complex activation cascade of the Orai1 channel. Under physiological conditions, Orai1 activation involves all four transmembrane (TM) domains and follows a sophisticated interaction mechanism that ensures accurate signal transmission from the protein periphery toward its central Ca2+-conducting pore. In this Review, we compile all currently known disease-associated Orai1 mutations, delineate the mechanisms by which they interfere with the activation cascade, and discuss their pathological relevance. Their widespread distribution across all the domains of this Ca2+ channel highlights that malfunctions at virtually any point along the Orai1 TM domain interfaces can profoundly impair its activation mechanism, ultimately leading to severe diseases.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 6","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-11-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148867956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions. 比率内质网钙传感器定量比较跨细胞类型和亚细胞区域。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-08-19 DOI: 10.1085/jgp.202413564

Ryan J Farrell, S Thomas Hennigan, Kirsten G Bredvik, Michael B Hoppa, Timothy A Brown, Timothy A Ryan

{"title":"A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.","authors":"Ryan J Farrell, S Thomas Hennigan, Kirsten G Bredvik, Michael B Hoppa, Timothy A Brown, Timothy A Ryan","doi":"10.1085/jgp.202413564","DOIUrl":"10.1085/jgp.202413564","url":null,"abstract":"The ER is an important regulator of Ca2+ in cells and dysregulation of ER Ca2+ homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impact cellular physiology would be facilitated by quantitative measurements of ER Ca2+ levels that allow for robust comparisons across conditions. To achieve this, we enhanced our original high dynamic range ER Ca2+ indicator, ER-GCaMP6-150, by fusing it to the HaloTag protein, which when bound to Janelia Fluor (JF) dyes creates a ratiometric ER Ca2+ probe. This probe (ER-Halo-GCaMP6-150) displayed minimal changes to the Ca2+-binding properties compared with our original ER Ca2+ probe as shown through in vitro and in cell Ca2+ calibrations. We describe a method to use this ratiometric probe for quantitative comparisons of ER Ca2+ concentrations and leverage this technique to compare ER Ca2+ levels across cell types and subcellular compartments. Using this approach, we show that the resting concentration of ER Ca2+ in primary dissociated neurons does not differ between excitatory and inhibitory subtypes nor between axonal and somatodendritic compartments. However, resting ER Ca2+ levels in neuronal somas are substantially lower than that measured in embryonic fibroblasts. The ER-Halo-GCaMP6-150 provides a robust tool to directly measure ER Ca2+ levels for studies of ER physiology across cell types and compartments.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148801767","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

ATP1A1 variants spring a leak. ATP1A1变异会导致泄漏。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-08-11 DOI: 10.1085/jgp.202614091

Ben Short

引用次数: 0

Characterization of an open-channel structure and lateral conduction pathway in the cation-selective pentameric ligand-gated ion channel, ELIC. 阳离子选择性五聚体配体门控离子通道（ELIC）中开放通道结构和横向传导途径的表征。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-08-27 DOI: 10.1085/jgp.202513926

Mark J Arcario, Elizabeth J Wu-Chen, Yuna Shim, Jérôme Hénin, Grace Brannigan, Wayland W L Cheng

{"title":"Characterization of an open-channel structure and lateral conduction pathway in the cation-selective pentameric ligand-gated ion channel, ELIC.","authors":"Mark J Arcario, Elizabeth J Wu-Chen, Yuna Shim, Jérôme Hénin, Grace Brannigan, Wayland W L Cheng","doi":"10.1085/jgp.202513926","DOIUrl":"10.1085/jgp.202513926","url":null,"abstract":"Open-channel structures of multiple pentameric ligand-gated ion channels (pLGICs) have been determined, including the prokaryotic model pLGIC, Erwinia ligand-gated ion channel (ELIC). For many of these structures, it remains uncertain whether they represent a physiologic open-channel state because the conditions used for structure determination do not match those of functional measurements in cell membranes. Here, MD simulation is used to examine the ion conduction properties of the ELIC open-channel structure, which was determined using a non-desensitizing mutant called ELIC5. Results from simulations show that the pore remains stably open on the microsecond timescale, but computational electrophysiology measurements demonstrate a large outward rectification and an inward conductance that is significantly lower than experiment. This discrepancy is attributed to a constricted extracellular domain (ECD), which restricts the passage of ions between the ECD vestibule and extracellular solution. Unbiased MD simulation of the ELIC5 structure demonstrates spontaneous widening of an intersubunit space in the ECD to expose a lateral fenestration, which becomes the dominant ion conduction pathway. Computational electrophysiology of the ELIC5 MD-refined structures with a widened lateral fenestration shows better agreement with experimental single-channel recordings. Mutations of residues along the lateral ion conduction pathway show reduced single-channel conductance, supporting the importance of the lateral fenestration for ion conduction in a cation-selective pLGIC.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13520879/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148834791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Locking the TMEM16A chloride channel into its conductive state. 将TMEM16A氯离子通道锁定到导电状态。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-07-30 DOI: 10.1085/jgp.202614052

Zhang Feng

引用次数: 0

Reorganization of the slide helix by cholesterol during KirBac1.1 channel inactivation in membranes. 在膜中KirBac1.1通道失活过程中胆固醇对载玻片螺旋的重组。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-08-06 DOI: 10.1085/jgp.202614000

Arpan Bysack, H Raghuraman

{"title":"Reorganization of the slide helix by cholesterol during KirBac1.1 channel inactivation in membranes.","authors":"Arpan Bysack, H Raghuraman","doi":"10.1085/jgp.202614000","DOIUrl":"https://doi.org/10.1085/jgp.202614000","url":null,"abstract":"KirBac1.1 is a prokaryotic homolog of mammalian inward-rectifier potassium (Kir) channels, and the functional equilibrium of KirBac1.1 is highly dependent on the type of membrane lipids. Like Kir channels, KirBac1.1 activity is also inhibited by cholesterol, a physiologically relevant lipid in human health and disease. Despite the slide helix is an important functional motif during lipid-dependent gating, the structural dynamics of the slide helix during cholesterol-induced channel inactivation is not well understood and is the focus of this work. Sequence analysis reveals that the slide helix of KirBac1.1 itself is a putative cholesterol-recognition motif. Liposomal K+ flux assays show that transport activity of the wild-type channel in PC/PG membranes is completely abolished at high concentration of cholesterol, whereas this is not observed in some of the single-cysteine mutants of the slide helix, indicating slide helix residues are critical for cholesterol sensitivity. Quenching of intrinsic Trp fluorescence upon increasing cholesterol concentration strongly suggests that the KirBac1.1 functional inhibition by cholesterol is possibly due to its direct interaction with the channel. Membrane penetration depth measurements using NBD-labeled slide helix residues clearly show relatively shallow membrane interfacial localization of the slide helix in cholesterol-containing membranes that is associated with significant structural dynamics changes and altered conformational heterogeneity. Based on distance measurements in varying membrane lipid compositions that stabilize the active and inactive conformations, we hypothesize that slide helix position in the membrane might possibly act as a \"conformational switch\" in regulating the KirBac1.1 function. These results involving dynamic lipid-protein interactions in lipid-dependent gating might be relevant for other Kir channels.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148681216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A TMEM63B variant with enhanced mechanosensitive channel activity and acquired lipid scramblase function. 一种TMEM63B变体，具有增强的机械敏感通道活性和获得的脂质超燃酶功能。

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-08-10 DOI: 10.1085/jgp.202614003

Augustus J Lowry, Pengfei Liang, Maria A Gonzalez Torres, Huanghe Yang

{"title":"A TMEM63B variant with enhanced mechanosensitive channel activity and acquired lipid scramblase function.","authors":"Augustus J Lowry, Pengfei Liang, Maria A Gonzalez Torres, Huanghe Yang","doi":"10.1085/jgp.202614003","DOIUrl":"10.1085/jgp.202614003","url":null,"abstract":"TMEM63B, a mechanosensitive ion channel (MSC), is associated with severe neurodevelopmental disorders, including severe early-onset developmental and epileptic encephalopathy. 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 cotransport. 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.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13519930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148702870","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

On the mechanism of hypomagnesemia with treatment-resistant seizures caused by variants of the Na+,K+-ATPase α1 subunit (ATP1A1). Na+，K+- atp酶α1亚基（ATP1A1）变异引起的低镁血症伴治疗抵抗性癫痫发作的机制

IF 3.2 2区医学

Journal of General Physiology Pub Date : 2026-09-07 Epub Date: 2026-06-30 DOI: 10.1085/jgp.202513959

Nicolas Colmano, Daniel Self, Hang N Nielsen, Rikke Holm, Chai C Gopalasingam, Ryota Fujimura, Kazuhiro Abe, Bente Vilsen, Sho T Yano, Pablo Artigas

{"title":"On the mechanism of hypomagnesemia with treatment-resistant seizures caused by variants of the Na+,K+-ATPase α1 subunit (ATP1A1).","authors":"Nicolas Colmano, Daniel Self, Hang N Nielsen, Rikke Holm, Chai C Gopalasingam, Ryota Fujimura, Kazuhiro Abe, Bente Vilsen, Sho T Yano, Pablo Artigas","doi":"10.1085/jgp.202513959","DOIUrl":"10.1085/jgp.202513959","url":null,"abstract":"Various dominant disease phenotypes are caused by germline variants of ATP1A1, the gene for the ubiquitously expressed Na+,K+-ATPase (NKA) α1 subunit. What characteristics link specific variants to a particular disease phenotype remains unclear. We used heterologous expression in mammalian cells and Xenopus oocytes to evaluate the characteristics of α1 variants causing hypomagnesemia with treatment-resistant seizures: L302R, G303R, M859R, and W931R. Cell survival in the presence of micromolar ouabain was reduced in cells expressing ouabain-resistant versions of these variants, indicating defective function of the variants relative to WT. Fluorescently tagged WT-α1 expressed in HEK293 cells showed plasmalemma localization, while all variants displayed increased cytosolic signal. In variant-expressing oocytes under voltage clamp, each variant showed passive \"leak\" currents, which were unaffected by ouabain in L302R and M859R, partially inhibited by ouabain in G303R and enhanced by ouabain in W931R. Na+ and Cl‒ permeate through G303R and W931R. M859R and W931R presented small K+-activated ouabain-inhibitable NKA currents that were absent in L302R and G303R. Growth of cells in ouabain and enzymatic studies demonstrated Na+/K+-dependent active transport for W931R, albeit with reduced Na+ affinity. We solved the cryo-EM structure of W931R-α1β1 with bound ouabain at 3 Å resolution. The structure showed a distorted transmembrane segment M9, which causes a concavity in the detergent micelle, suggesting that similar distortions may provide a leak pathway at the protein-lipid interface. Our results demonstrate that besides a loss of NKA function, all four variants carry an aberrant leak current that probably underlies the seizures and the hypomagnesemia characterizing this phenotype.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 5","pages":""},"PeriodicalIF":3.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13317510/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148354698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0