Journal of General Physiology最新文献_第3页

Fluorescence labeling strategies for cell surface expression of TRPV1. 细胞表面表达 TRPV1 的荧光标记策略。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-08-20 DOI: 10.1085/jgp.202313523

Taylor M Mott, Grace C Wulffraat, Alex J Eddins, Ryan A Mehl, Eric N Senning

{"title":"Fluorescence labeling strategies for cell surface expression of TRPV1.","authors":"Taylor M Mott, Grace C Wulffraat, Alex J Eddins, Ryan A Mehl, Eric N Senning","doi":"10.1085/jgp.202313523","DOIUrl":"10.1085/jgp.202313523","url":null,"abstract":"Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a trans-cyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11338283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142005960","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

Rad protein: An essential player in L-type Ca2+ channel localization and modulation in cardiomyocytes. Rad 蛋白：心肌细胞中 L 型 Ca2+ 通道定位和调节的重要参与者。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-08-22 DOI: 10.1085/jgp.202413629

Cherrie H T Kong, Eef Dries

引用次数: 0

Ion channels of cold transduction and transmission. 冷传导和传输的离子通道。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-07-25 DOI: 10.1085/jgp.202313529

Cheyanne M Lewis, Theanne N Griffith

{"title":"Ion channels of cold transduction and transmission.","authors":"Cheyanne M Lewis, Theanne N Griffith","doi":"10.1085/jgp.202313529","DOIUrl":"10.1085/jgp.202313529","url":null,"abstract":"Thermosensation requires the activation of a unique collection of ion channels and receptors that work in concert to transmit thermal information. It is widely accepted that transient receptor potential melastatin 8 (TRPM8) activation is required for normal cold sensing; however, recent studies have illuminated major roles for other ion channels in this important somatic sensation. In addition to TRPM8, other TRP channels have been reported to contribute to cold transduction mechanisms in diverse sensory neuron populations, with both leak- and voltage-gated channels being identified for their role in the transmission of cold signals. Whether the same channels that contribute to physiological cold sensing also mediate noxious cold signaling remains unclear; however, recent work has found a conserved role for the kainite receptor, GluK2, in noxious cold sensing across species. Additionally, cold-sensing neurons likely engage in functional crosstalk with nociceptors to give rise to cold pain. This Review will provide an update on our understanding of the relationship between various ion channels in the transduction and transmission of cold and highlight areas where further investigation is required.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11273221/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141762722","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

Novel cardiac myosin inhibitor for hypertrophic cardiomyopathy. 治疗肥厚型心肌病的新型心肌酶抑制剂。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-08-12 DOI: 10.1085/jgp.202413640

Danuta Szczesna-Cordary

引用次数: 0

Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor. 新型调节轻链依赖性心肌肌球蛋白抑制剂的作用机制

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-07-31 DOI: 10.1085/jgp.202313503

Kristina Kooiker, Qing-Fen Gan, Ming Yu, Na Sa, Saffie Mohran, Yuanhua Cheng, Galina Flint, Stephanie Neys, Chengqian Gao, Devin Nissen, Tim McMillen, Anthony Asencio, Weikang Ma, Thomas C Irving, Farid Moussavi-Harami, Michael Regnier

{"title":"Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor.","authors":"Kristina Kooiker, Qing-Fen Gan, Ming Yu, Na Sa, Saffie Mohran, Yuanhua Cheng, Galina Flint, Stephanie Neys, Chengqian Gao, Devin Nissen, Tim McMillen, Anthony Asencio, Weikang Ma, Thomas C Irving, Farid Moussavi-Harami, Michael Regnier","doi":"10.1085/jgp.202313503","DOIUrl":"10.1085/jgp.202313503","url":null,"abstract":"Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11291911/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141857225","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

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents. ω-Grammotoxin-SIA抑制电压门控Na+通道电流

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-07-23 DOI: 10.1085/jgp.202413563

Rita de Cássia Collaço, Filip Van Petegem, Frank Bosmans

{"title":"ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.","authors":"Rita de Cássia Collaço, Filip Van Petegem, Frank Bosmans","doi":"10.1085/jgp.202413563","DOIUrl":"10.1085/jgp.202413563","url":null,"abstract":"ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141749788","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

Using neural networks for image analysis in general physiology. 利用神经网络进行普通生理学图像分析。

IF 3.8 2区医学

Journal of General Physiology Pub Date : 2024-09-17 DOI: 10.1085/jgp.202413623

Eduardo Rios

引用次数: 0

Artificial intelligence approaches to the volumetric quantification of glycogen granules in EM images of human tissue. 用人工智能方法对人体组织电磁图像中的糖原颗粒进行体积量化。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-09-02 Epub Date: 2024-07-09 DOI: 10.1085/jgp.202413595

Eduardo Ríos, Montserrat Samsó, Lourdes C Figueroa, Carlo Manno, Eshwar R Tammineni, Lucas Rios Giordano, Sheila Riazi

{"title":"Artificial intelligence approaches to the volumetric quantification of glycogen granules in EM images of human tissue.","authors":"Eduardo Ríos, Montserrat Samsó, Lourdes C Figueroa, Carlo Manno, Eshwar R Tammineni, Lucas Rios Giordano, Sheila Riazi","doi":"10.1085/jgp.202413595","DOIUrl":"10.1085/jgp.202413595","url":null,"abstract":"Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle. Two UNet-type semantic segmentation models were built: \"Locations\" classified pixels as belonging to different regions in the cell; \"Granules\" identified pixels within granules. From their joint output, a pixel fraction pf was calculated for images from patients positive (MHS) or negative (MHN) to a test for malignant hyperthermia susceptibility. pf was used to derive vf, the volume fraction occupied by granules. The relationship vf (pf) was derived from a simulation of volumes (\"baskets\") containing virtual granules at realistic concentrations. The simulated granules had diameters matching the real ones, which were measured by adapting a utility devised for calcium sparks. Applying this relationship to the pf measured in images, vf was calculated for every region and patient, and from them a glycogen concentration. The intermyofibrillar spaces and the sarcomeric I band had the highest granular content. The measured glycogen concentration was low enough to allow for a substantial presence of non-granular glycogen. The MHS samples had an approximately threefold lower concentration (significant in a hierarchical test), consistent with earlier evidence of diminished glucose processing in MHS. The AI models and the approach to infer three-dimensional magnitudes from two-dimensional images should be adaptable to other tasks on a variety of images from patients and animal models and different disease conditions.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 9","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11233403/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141560377","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

Beyond homogenates: New tool available for estimating glycogen's numerical subcellular distribution. 超越匀浆：估算糖原亚细胞分布的新工具。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-09-02 Epub Date: 2024-07-09 DOI: 10.1085/jgp.202413607

Joachim Nielsen

引用次数: 0

The C-terminus of Rad is required for membrane localization and L-type calcium channel regulation. Rad 的 C 端是膜定位和 L 型钙通道调节所必需的。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-09-02 Epub Date: 2024-07-11 DOI: 10.1085/jgp.202313518

Garrett Elmore, Brooke M Ahern, Nicholas M McVay, Kyle W Barker, Sarisha S Lohano, Nemat Ali, Andrea Sebastian, Douglas A Andres, Jonathan Satin, Bryana M Levitan

{"title":"The C-terminus of Rad is required for membrane localization and L-type calcium channel regulation.","authors":"Garrett Elmore, Brooke M Ahern, Nicholas M McVay, Kyle W Barker, Sarisha S Lohano, Nemat Ali, Andrea Sebastian, Douglas A Andres, Jonathan Satin, Bryana M Levitan","doi":"10.1085/jgp.202313518","DOIUrl":"10.1085/jgp.202313518","url":null,"abstract":"L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel β subunits (CaVβ) to promote inhibition of ICa,L. In addition to CaVβ interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to β-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 9","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11244639/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141581549","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