Journal of General Physiology最新文献

{"title":"Beat-locked ATP microdomains in the sinoatrial node map a Ca2+-timed energetic hierarchy and regional pacemaker roles.","authors":"Manuel F Muñoz, Collin Matsumoto, Paula Rhana, Declan Manning, Geoanna M Bautista, Daniel M Collier, L Fernando Santana","doi":"10.1085/jgp.202513874","DOIUrl":"10.1085/jgp.202513874","url":null,"abstract":"<p><p>Pacemaker cells of the sinoatrial (SA) node fire spontaneously and continuously under normal conditions, sustaining a high energetic cost with every heartbeat. How they meet this demand has remained poorly understood. Using genetically encoded fluorescent sensors targeted to the cytosol and mitochondria, we tracked ATP in real time within myocytes of the mouse SA node. Rather than maintaining a steady energy reserve, these cells produce and consume ATP in precise, beat-by-beat bursts in both compartments, synchronized to each Ca2+ transient that triggers a heartbeat-a just-in-time energetic strategy. Not all pacemaker cells operate equally. Cells in the superior SA node, better supplied by blood vessels and rich in mitochondria, produce ATP more efficiently with each beat-a high-gain phenotype. Cells in the inferior node, more sparsely vascularized, operate in a lower gain or energy-deficit mode. These distinct energetic profiles set limits on the firing frequencies each cell can sustain, determining which cells drive fast rates and which support stable rhythm across a broader frequency range. Blocking Ca2+ transfer into mitochondria or impairing ATP export abolished beat-locked energy signals, consistent with mitochondrial Ca2+ uniporter-ANT machinery coupling Ca2+ release to ATP fluctuations. Strikingly, disrupting mitochondrial energy production rendered pacemaker cells electrically silent, suggesting that mitochondrial ATP synthesis is essential for excitability. Together, these findings suggest that beat-locked, just-in-time ATP generation is integral to cardiac pacemaking itself, with local blood supply, mitochondrial capacity, and Ca2+ signaling shaping which cells preferentially set heart rate and which support stable firing across a broader frequency range.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13037587/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147582926","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}

{"title":"Opening of rod photoreceptor CNG channels by photodynamically generated singlet oxygen.","authors":"Gaogao He, Xiaoxi Li, Yanlin Huang, Conghan Wang, Minjie Tan, Qinglian Liu, Lei Zhou","doi":"10.1085/jgp.202513819","DOIUrl":"10.1085/jgp.202513819","url":null,"abstract":"<p><p>Molecular oxygen exists in three electronic states: the triplet ground state and two singlet (1O2) excited states. Under physiological conditions, 1O2 can be produced either through photodynamic processes, which require light, photosensitizer, and oxygen, or via metabolic reactions involving enzymes and other reactive oxygen species (ROS). 1O2 readily reacts with biomacromolecules, however, its volatile chemical nature and the lack of precise working models hamper the study of its molecular mechanism and physiological significance. Here we report that human CNG channels from rod photoreceptors are very sensitive to the process of photodynamic modification (PDM). Multiple lines of evidence indicate 1O2 is the major player in PDM, including the application of a genetically encoded photosensitizer, a popularly used photosensitizer to produce 1O2, and two known quenchers for 1O2. The 1O2-mediated modification increases the opening of hCNGA1 in the absence or under subsaturating concentrations of cyclic guanosine monophosphate (cGMP), and in conjunction with ligand gating, acts synergistically on channel opening. Mutagenesis and mass spectroscopy (MS) analysis reveal key residues affecting the PDM process. Taken together, through tackling the PDM of rod photoreceptor CNG channels, this study provides essential insights into the modification of protein molecules by 1O2, a ubiquitous and potentially critical signaling molecule.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147357898","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}

{"title":"Calmodulinopathy variants impair CaV1.3 and CaV2.1 regulation.","authors":"John W Hussey, Emily DeMarco, Deborah DiSilvestre, Malene Brohus, Ana-Octavia Busuioc, Emil D Iversen, Helene H Jensen, Mette Nyegaard, Michael T Overgaard, Manu Ben-Johny, Ivy E Dick","doi":"10.1085/jgp.202413734","DOIUrl":"https://doi.org/10.1085/jgp.202413734","url":null,"abstract":"<p><p>Calmodulinopathies are caused by mutations in calmodulin (CaM) and result in debilitating cardiac arrythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In addition, many patients exhibit neurological comorbidities, including developmental delay and autism spectrum disorder. Prior studies have identified the impairment of Ca2+/CaM-dependent inactivation (CDI) of CaV1.2 channels as a major pathogenic mechanism leading to the LQTS phenotype in these patients. However, the impact of these mutations on other voltage-gated calcium channels (VGCCs) has yet to be fully explored. Here, we examine the potential for pathological CaM variants to impair the Ca2+/CaM-dependent regulation of CaV1.3 and CaV2.1, both essential for neuronal function. We find that pathogenic mutations in CaM can impair the CDI of CaV1.3, with overlapping yet distinct disruption of the Ca2+-dependent facilitation (CDF) of CaV2.1 channels. Moreover, while the majority of CaM variants demonstrated the ability to bind the IQ region of each channel, differences were noted between CaV1.3 and CaV2.1, demonstrating unique CaM interactions across the two channel subtypes. Further, C-lobe CaM variants display a reduced ability to sense Ca2+ when in complex with the CaV IQ domains, explaining the Ca2+/CaM regulation deficits. Overall, these results support the possibility that disrupted Ca2+/CaM regulation of multiple VGCCs may contribute to the pathogenesis of calmodulinopathies.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147789738","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}

{"title":"Combining elastic network models and linear response theory as tool to understand the global dynamics in allosteric regulation of HCN channels.","authors":"Magnus Behringer, Jan Krumbach, Alessandro Porro, Andrea Saponaro, Dario DiFrancesco, Anna Moroni, Kay Hamacher, Gerhard Thiel","doi":"10.1085/jgp.202513899","DOIUrl":"10.1085/jgp.202513899","url":null,"abstract":"<p><p>Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) play important regulatory roles in the heart and the brain. At the core of their physiological functions is an activation by negative membrane potential and its modulation by cyclic nucleotides. While recent high-resolution cryo-EM structures combined with MD simulations have provided insights into fast events in the pore, like ion permeation, block, and cation-selectivity, the mechanism of slow allosteric regulation of gating by voltage and cyclic nucleotides remains poorly understood. Since slow conformational changes in proteins are largely determined by their global dynamics, coarse-grained computational methods such as elastic network models (ENMs) and linear response theory (LRT) analyses have been used to elucidate the intrinsic collective dynamics in HCN proteins associated with cyclic nucleotide-modulated gating. In this overview, we demonstrate the good performance of coarse-grained methods in predicting long-range conformational changes in HCN channels with respect to experimentally determined conformational states in these proteins with and without bound ligand. This provides general insights into the mechanical coupling of domains in HCN channels and on how their general tectonics enables bidirectional modulation between the binding site for cyclic nucleotides in the cytosol and the distant voltage-sensitive domain in the plasma membrane-embedded part of the protein.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147629162","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}

{"title":"Post-translational modifications of cardiac myosin-binding protein-C: Mechanisms behind fine-tuning the sarcomere.","authors":"Angela C Greenman, Willem de Lange, J Carter Ralphe","doi":"10.1085/jgp.202513889","DOIUrl":"10.1085/jgp.202513889","url":null,"abstract":"<p><p>Since the discovery of the cardiac isoform of myosin-binding protein-C (cMyBP-C), there has been continued interest in how cMyBP-C impacts cardiac function in both health and disease. cMyBP-C is a regulatory protein in the sarcomere that controls beat-to-beat changes in contractility in response to dynamic environmental demands placed upon the heart. Changes in force production during the contractile cycle are modulated through interactions of cMyBP-C with myosin and actin. Post-translational modifications (PTMs) of cMyBP-C, of which phosphorylation has received the most attention, are critical to the function of cMyBP-C in the healthy heart and is affected in many disease states. While each of the PTMs that will be discussed in this review have known and often widespread effects on important cellular processes spanning transcriptional regulation, cell signaling, and metabolism, their impact on cMyBP-C function remains poorly understood and in some cases unverified. This Review focuses on the current understanding of cMyBP-C PTMs, namely phosphorylation, S-glutathionylation, S-nitrosylation, acetylation, citrullination, carbonylation, and O-GlcNAcylation. The potential for PTMs to exert wide ranging and likely nuanced effects may influence the range of cMyBP-C's response to varied conditions and may offer opportunities to identify novel therapeutic paradigms in the setting of disease.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147345874","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}

{"title":"Titin's P-zone domains A164-167 are essential for thick filament structural arrangement.","authors":"Catherine Hoover Browne, Seong-Won Han, Gerrie P Farman, John E Smith, Justin Kolb, Jochen Gohlke, Paul R Langlais, Paola Tonino, Mei Methawasin, Robbert van der Pijl, Henk Granzier","doi":"10.1085/jgp.202513891","DOIUrl":"10.1085/jgp.202513891","url":null,"abstract":"<p><p>The sarcomeric protein titin plays a central role in thick filament structure and function through its modular A-band domains, including the understudied P-zone, which links the C-zone to the M-band. To investigate the first four domains of titin's P-zone (A164-A167), we deleted them in a mouse model (TtnΔA164-167). Echocardiography and cardiomyocyte mechanics revealed mild changes to diastolic function and enlargement of the heart, but preserved contractility. The EDL muscle showed contractile deficits at the whole muscle level and increased passive stiffness at the myofiber level. Immunoelectron and super-resolution microscopy revealed altered thick filament architecture, including a ∼40-nm shift of titin and myosin binding protein-C epitopes toward the M-band, disruption of titin's α and β conformations, and shorter thick filaments. The structural changes are consistent with the loss of a myosin helical repeat. These findings establish a key structural role of titin's P-zone domains A164-A167 in templating thick filament protein arrangement, including the importance of titin's α and β conformations.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 3","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12981300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147437711","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}

{"title":"A primer on the methods of skeletal and cardiac muscle mechanics using permeabilized preparations.","authors":"Anthony L Hessel, Katelyn M Manross, Matthew M Borkowski, Christopher D Rand, Khoi Nguyen","doi":"10.1085/jgp.202513773","DOIUrl":"10.1085/jgp.202513773","url":null,"abstract":"<p><p>Permeabilized muscle fibers have a chemically disturbed sarcolemma that allows for the mixing of the extra- and intracellular environments and is important for a large variety of experimental methods. The experimental tools and skillsets used to study muscle mechanics vary widely between groups and are often underreported in published methodologies. More accessible details help improve the transparency of the method and provide primary reference material. To that end, we use our firsthand experiences to provide a guide for the preparation and use of permeabilized fibers. We focus on tissue collection, experimental apparatus design and function, practical considerations for handling preparations during an experiment, and detail some key changes to the structure of permeabilized samples. We further suggest ways scientists can take advantage of emerging technologies to increase experimental throughput, decrease experimental error, and support (or improve) data quality.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 2","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13064817/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146068998","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}

{"title":"The relationship between apparent potentiation and the magnitude of the control response.","authors":"Joe Henry Steinbach, Gustav Akk","doi":"10.1085/jgp.202513894","DOIUrl":"10.1085/jgp.202513894","url":null,"abstract":"<p><p>The effect of a potentiating drug on ion channel function is typically evaluated by comparing current responses to the control agonist in the presence and absence of the potentiator. Differences in ratios of responses are then taken as proof of distinct potentiation properties when comparing modulation by different compounds. In these experiments, the concentration of the agonist is typically kept low to generate a small fractional control response. The precise relative magnitude of the control response is, however, not standardized among labs and can range from a concentration producing a response equal to just 2% of maximal (EC2) to over EC25 in different studies. Here, we have investigated the relationship between the magnitude of the control response and the expected response ratio. As the EC value of the control response increases, the ratio of responses to agonist in the presence and absence of the potentiator decreases. We provide equations to calculate the expected response ratios at different levels of control responses and free energy changes at different response ratios. Lastly, we discuss the effect of the value of EC of the control response on the efficacy of negative allosteric inhibitors.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 2","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146055246","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}

{"title":"Molecular dynamics analyses of CLDN15 pore size and charge selectivity.","authors":"Sarah McGuinness, Pan Li, Ye Li, Shadi Fuladi, Sukanya Konar, Samaneh Sajjadi, Mohammed Sidahmed, Yueying Li, Le Shen, Fatemeh Khalili-Araghi, Christopher R Weber","doi":"10.1085/jgp.202513824","DOIUrl":"10.1085/jgp.202513824","url":null,"abstract":"<p><p>Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and an all-atom molecular dynamics simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid or neutral amino acid asparagine, our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes nonessential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggest that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 2","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12898017/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146168113","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}

{"title":"Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence.","authors":"Thierry Cens, Matthieu Rousset, Claudine Menard, Mohamed Chahine, Claude Collet, Jean-Christophe Sandoz, Alain Chavanieu, Sebastien Estaran, Jean-Baptiste Thibaud, Patrick Bois, Pierre Charnet","doi":"10.1085/jgp.202413712","DOIUrl":"10.1085/jgp.202413712","url":null,"abstract":"<p><p>The recently characterized honeybee CaV4 channel is a high-voltage-activated Ca2+ channel ortholog to the DSC1 channel identified in Drosophila. While sequence similarities to NaV channels are obvious, permeation properties and current kinetics are more closely aligned with those of CaV channels. CaV4 exhibits a distinctive cation-dependent inactivation pattern, a hallmark of Ca2+ channel behavior, and nonetheless displays sensitivity to a Na+ channel-specific regulator, veratrine. Calcium channel facilitation is a phenomenon whereby the probability of calcium channel opening increases with successive depolarization pulses, resulting in an enhanced Ca2+ influx during repetitive or sustained electrical activity. In this study, we have identified an additional specific property of CaV4 in the form of an atypical voltage-dependent facilitation of the Ca2+ or Ba2+ currents by strong pre-depolarizations or prepulses (pPs). This physiologically relevant phenomenon, known as pP-induced facilitation (PiF), is subject to positive regulation by the amplitude of the pP but to negative regulation by its duration. It produces a hyperpolarizing shift of the I-V curve without any change in the reversal potential and macroscopic or single channel conductance. PiF is thus more pronounced for small depolarizations and almost absent when channels reach their maximal open probability. A mutation that affects the inactivation of the CaV4 channel prevents the occurrence of PiF. This previously undocumented form of facilitation appears exclusive to CaV4 channels. A strong pP may lock CaV4 channels in a pre-open state, rendering them more susceptible to activation and thereby shifting the activation curve toward more negative potentials. This, in turn, would accelerate channel opening and increase current amplitude. Lastly, we show that the inactivation particle of CaV4 (MFLT sequence, equivalent to the IFMT motif in human NaV, or MFMT in Apis NaV channel), in addition to its role in the initiation of the voltage-dependent inactivation, also modulates PiF.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 2","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146151371","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}