Journal of General Physiology最新文献_第9页

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

Epilepsy-associated Kv1.1 channel subunits regulate intrinsic cardiac pacemaking in mice. 癫痫相关 Kv1.1 通道亚基调节小鼠固有的心脏起搏。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-09-02 Epub Date: 2024-07-22 DOI: 10.1085/jgp.202413578

Man Si, Ahmad Darvish, Kelsey Paulhus, Praveen Kumar, Kathryn A Hamilton, Edward Glasscock

{"title":"Epilepsy-associated Kv1.1 channel subunits regulate intrinsic cardiac pacemaking in mice.","authors":"Man Si, Ahmad Darvish, Kelsey Paulhus, Praveen Kumar, Kathryn A Hamilton, Edward Glasscock","doi":"10.1085/jgp.202413578","DOIUrl":"10.1085/jgp.202413578","url":null,"abstract":"The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking.","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/PMC11261506/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141735693","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

Kv1.1 channels help set the pace. Kv1.1 通道有助于确定节奏。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-09-02 Epub Date: 2024-08-07 DOI: 10.1085/jgp.202413649

Ben Short

引用次数: 0

ON and OFF starburst amacrine cells are controlled by distinct cholinergic pathways. 星爆肾上腺素细胞的 ON 和 OFF 受控于不同的胆碱能通路。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-08-05 Epub Date: 2024-06-05 DOI: 10.1085/jgp.202413550

Mie Gangi, Takuma Maruyama, Toshiyuki Ishii, Makoto Kaneda

{"title":"ON and OFF starburst amacrine cells are controlled by distinct cholinergic pathways.","authors":"Mie Gangi, Takuma Maruyama, Toshiyuki Ishii, Makoto Kaneda","doi":"10.1085/jgp.202413550","DOIUrl":"10.1085/jgp.202413550","url":null,"abstract":"Cholinergic signaling in the retina is mediated by acetylcholine (ACh) released from starburst amacrine cells (SACs), which are key neurons for motion detection. SACs comprise ON and OFF subtypes, which morphologically show mirror symmetry to each other. Although many physiological studies on SACs have targeted ON cells only, the synaptic computation of ON and OFF SACs is assumed to be similar. Recent studies demonstrated that gene expression patterns and receptor types differed between ON and OFF SACs, suggesting differences in their functions. Here, we compared cholinergic signaling pathways between ON and OFF SACs in the mouse retina using the patch clamp technique. The application of ACh increased GABAergic feedback, observed as postsynaptic currents to SACs, in both ON and OFF SACs; however, the mode of GABAergic feedback differed. Nicotinic receptors mediated GABAergic feedback in both ON and OFF SACs, while muscarinic receptors mediated GABAergic feedback in ON SACs only in adults. Neither tetrodotoxin, which blocked action potentials, nor LY354740, which blocked neurotransmitter release from SACs, eliminated ACh-induced GABAergic feedback in SACs. These results suggest that ACh-induced GABAergic feedback in ON and OFF SACs is regulated by different feedback mechanisms in adults and mediated by non-spiking amacrine cells other than SACs.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11153316/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141249087","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

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement. 不同的荧光标签报告了 spHCN 通道电压传感器运动的不同组成部分。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-08-05 Epub Date: 2024-07-05 DOI: 10.1085/jgp.202413559

Magdalena N Wojciechowski, Chaseley E McKenzie, Andrew Hung, Alibek Kuanyshbek, Ming S Soh, Christopher A Reid, Ian C Forster

{"title":"Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.","authors":"Magdalena N Wojciechowski, Chaseley E McKenzie, Andrew Hung, Alibek Kuanyshbek, Ming S Soh, Christopher A Reid, Ian C Forster","doi":"10.1085/jgp.202413559","DOIUrl":"10.1085/jgp.202413559","url":null,"abstract":"We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11223168/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141538955","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

Fusion pore flux controls the rise-times of quantal synaptic responses. 融合孔通量控制着量子突触反应的上升时间。

IF 3.3 2区医学

Journal of General Physiology Pub Date : 2024-08-05 Epub Date: 2024-06-11 DOI: 10.1085/jgp.202313484

Meyer B Jackson, Chung-Wei Chiang, Jinbo Cheng

{"title":"Fusion pore flux controls the rise-times of quantal synaptic responses.","authors":"Meyer B Jackson, Chung-Wei Chiang, Jinbo Cheng","doi":"10.1085/jgp.202313484","DOIUrl":"10.1085/jgp.202313484","url":null,"abstract":"The release of neurotransmitter from a single synaptic vesicle generates a quantal response, which at excitatory synapses in voltage-clamped neurons is referred to as a miniature excitatory postsynaptic current (mEPSC). We analyzed mEPSCs in cultured mouse hippocampal neurons and in HEK cells expressing postsynaptic proteins enabling them to receive synaptic inputs from cocultured neurons. mEPSC amplitudes and rise-times varied widely within and between cells. In neurons, mEPSCs with larger amplitudes had longer rise-times, and this correlation was stronger in neurons with longer mean rise-times. In HEK cells, this correlation was weak and unclear. Standard mechanisms thought to govern mEPSCs cannot account for these results. We therefore developed models to simulate mEPSCs and assess their dependence on different factors. Modeling indicated that longer diffusion times for transmitters released by larger vesicles to reach more distal receptors cannot account for the correlation between rise-time and amplitude. By contrast, incorporating the vesicle size dependence of fusion pore expulsion time recapitulated experimental results well. Larger vesicles produce mEPSCs with larger amplitudes and also take more time to lose their content. Thus, fusion pore flux directly contributes to mEPSC rise-time. Variations in fusion pores account for differences among neurons, between neurons and HEK cells, and the correlation between rise-time and the slope of rise-time versus amplitude plots. Plots of mEPSC amplitude versus rise-time are sensitive to otherwise inaccessible properties of a synapse and offer investigators a means of assessing the role of fusion pores in synaptic release.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"156 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11167452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141302127","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