Journal of General Physiology最新文献_第4页

On the mechanism of K+ transport through the inter-subunit tunnel of KdpFABC. KdpFABC亚基间通道中K+输运机制的研究。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-12 DOI: 10.1085/jgp.202513794

Hridya Valia Madapally, Adel Hussein, Martin Wazar Eriksen, Bjørn Panyella Pedersen, David L Stokes, Himanshu Khandelia

{"title":"On the mechanism of K+ transport through the inter-subunit tunnel of KdpFABC.","authors":"Hridya Valia Madapally, Adel Hussein, Martin Wazar Eriksen, Bjørn Panyella Pedersen, David L Stokes, Himanshu Khandelia","doi":"10.1085/jgp.202513794","DOIUrl":"10.1085/jgp.202513794","url":null,"abstract":"KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12833782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145745833","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

Embedding muscle fibers in hydrogel improves viability and preserves contractile function during prolonged ex vivo culture. 在长时间的离体培养中，将肌肉纤维包埋在水凝胶中可以提高活力并保持收缩功能。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-11-03 DOI: 10.1085/jgp.202513761

Leander A Vonk, Osman Esen, Daan Hoomoedt, Rajvi M N Balesar, Coen A C Ottenheijm, Tyler J Kirby

{"title":"Embedding muscle fibers in hydrogel improves viability and preserves contractile function during prolonged ex vivo culture.","authors":"Leander A Vonk, Osman Esen, Daan Hoomoedt, Rajvi M N Balesar, Coen A C Ottenheijm, Tyler J Kirby","doi":"10.1085/jgp.202513761","DOIUrl":"10.1085/jgp.202513761","url":null,"abstract":"Ex vivo culture of isolated muscle fibers can serve as an important model for in vitro research on mature skeletal muscle fibers. Nevertheless, this model has limitations for long-term studies due to structural loss and dedifferentiation following prolonged culture periods. This study aimed to investigate how ex vivo culture affects muscle fiber contraction and to improve the culture system to preserve muscle fiber morphology and sarcomere function. Additionally, we sought to determine which culture-induced changes can negatively affect muscle fiber contraction. We cultured isolated flexor digitorum brevis (FDB) muscle fibers in several conditions for up to 7 days and investigated viability, morphology, and the unloaded sarcomere shortening in intact fibers, along with force generation in permeabilized muscle fibers. In addition, we examined changes to the microtubule network. We found a time-dependent decrease in contractility and viability in muscle fibers cultured for 7 days on a laminin-coated culture dish (2D). Conversely, we found that culturing FDB muscle fibers in a low-serum, fibrin/Geltrex hydrogel (3D) reduces markers of muscle fiber dedifferentiation (i.e., sprouting), improves viability, and retains contractility over time. We discovered that the loss of contractility of cultured muscle fibers was not the direct result of reduced sarcomere function but may be related to changes in the microtubule network. Collectively, our findings highlight the importance of providing muscle fibers with a 3D environment during ex vivo culture, particularly when testing pharmacological or genetic interventions to study viability or contractile function.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12581944/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145440150","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 binding mode for negative allosteric NMDA receptor modulators. 负变构NMDA受体调节剂的新结合模式。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-03 DOI: 10.1085/jgp.202513872

James S Lotti, Jed T Syrenne, Avery J Benton, Ahmad Al-Mousawi, Lauren E Cornelison, Christopher J Trolinder, Feng Yi, Zhucheng Zhang, Cindee K Yates-Hansen, Levi J McClelland, James Bosco, Andrew R Rau, Rasmus P Clausen, Kasper B Hansen

{"title":"Novel binding mode for negative allosteric NMDA receptor modulators.","authors":"James S Lotti, Jed T Syrenne, Avery J Benton, Ahmad Al-Mousawi, Lauren E Cornelison, Christopher J Trolinder, Feng Yi, Zhucheng Zhang, Cindee K Yates-Hansen, Levi J McClelland, James Bosco, Andrew R Rau, Rasmus P Clausen, Kasper B Hansen","doi":"10.1085/jgp.202513872","DOIUrl":"10.1085/jgp.202513872","url":null,"abstract":"NMDA-type ionotropic glutamate receptors mediate excitatory neurotransmission and synaptic plasticity, but aberrant signaling by these receptors is also implicated in brain disorders. Here, we present the binding site and the mechanism of action for UCM-101, a novel negative NMDA receptor modulator that produces full inhibition of NMDA receptor-mediated excitatory postsynaptic currents in hippocampal CA pyramidal neurons from juvenile mouse brain slices. UCM-101 has a 59-fold higher binding affinity at GluN1/2A compared with GluN1/2B receptors and inhibits diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors with IC50 values of 110 and 240 nM, respectively, in the presence of 1 µM glycine. The novel binding mode for UCM-101 is revealed in a high-resolution crystal structure of the GluN1/2A agonist binding domain heterodimer. UCM-101 and its analog TCN-213 inhibit NMDA receptors by negatively modulating co-agonist binding to the GluN1 subunit via an allosteric mechanism that is conserved with previously described GluN2A-selective antagonists, TCN-201 and MPX-004. Despite the shared mechanism of action, the structural determinants that mediate subunit selectivity for UCM-101 are distinct from those of TCN-201 and MPX-004. These findings provide detailed insights into the binding site and mechanism of action of a novel NMDA receptor modulator and open new avenues for the development of NMDA receptor ligands with therapeutic potential.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12772459/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145670558","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

Cavβ dances the two-step with VSD II. Cavβ与VSD II跳两步舞。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-18 DOI: 10.1085/jgp.202513935

Ben Short

引用次数: 0

Optical control of PI(4,5)P2 sensitivity of ion channels by manipulation of single lysine residue. 单赖氨酸残基对离子通道PI(4,5)P2灵敏度的光学控制。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-11-18 DOI: 10.1085/jgp.202513811

Junxian Zhou, Rizki Tsari Andriani, Natsuki Mizutani, Kohei Yamamoto, Daisuke Yoshioka, Akira Kawanabe, Takafumi Kawai, Yoshifumi Okochi, Yasushi Okamura

{"title":"Optical control of PI(4,5)P2 sensitivity of ion channels by manipulation of single lysine residue.","authors":"Junxian Zhou, Rizki Tsari Andriani, Natsuki Mizutani, Kohei Yamamoto, Daisuke Yoshioka, Akira Kawanabe, Takafumi Kawai, Yoshifumi Okochi, Yasushi Okamura","doi":"10.1085/jgp.202513811","DOIUrl":"10.1085/jgp.202513811","url":null,"abstract":"Polyphosphoinositides (PPIns) are essential components of membrane lipids and play crucial roles in cell signaling in eukaryotes. Phosphatidylinositol4,5-bisphosphate (PI(4,5)P2) is a species of PPIns enriched in the plasma membrane and regulates numerous membrane proteins, including ion channels, transporters, and receptors, primarily through direct binding to positively charged residues such as lysine and arginine. Despite recent advances in structural biology and biophysics, the specific contributions of individual amino acid residues to PI(4,5)P2 binding in membrane proteins remain unclear. These questions have been explored by functional characterization of mutant proteins with site-specific amino acid replacement and their comparison with the WT proteins. Here, we apply genetic code expansion to investigate the role of lysine residues in the PI(4,5)P2 sensitivity of ion channels. A caged lysine compound, hydroxycoumarin-lysine (HCK), was incorporated at several key lysine residues critical for PI(4,5)P2 sensitivity in the mouse inward-rectifier potassium channel Kir2.1, expressed in Xenopus oocytes. Caging of lysine by introducing HCK at K182 or K187 completely silenced Kir2.1 currents, but light-induced uncaging restored current activity. Voltage-sensing phosphatase assays revealed that this current increase was accompanied by enhanced PI(4,5)P2 sensitivity. On the other hand, introducing HCK at K219, which forms a secondary PI(4,5)P2-binding region, did not fully eliminate Kir2.1 currents, and uncaging resulted in an approximately twofold increase in current. Analysis of uncaging and PI(4,5)P2 sensitivity in Kir2.1-K219HCK revealed that the region C-terminal to residue K219 is dispensable when assembled with the full-length protein. Genetic code expansion using caged lysine provides a valuable tool for studying the mechanisms of PI(4,5)P2 regulation in ion channels, complementing existing approaches.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145544153","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

Subtype-selective targeting of NMDA receptors-A potent new compound emerges. 亚型选择性靶向NMDA受体-一种有效的新化合物出现。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-03 DOI: 10.1085/jgp.202513920

Hiro Furukawa

{"title":"Subtype-selective targeting of NMDA receptors-A potent new compound emerges.","authors":"Hiro Furukawa","doi":"10.1085/jgp.202513920","DOIUrl":"10.1085/jgp.202513920","url":null,"abstract":"Subtype-selective modulation of N-methyl-D-aspartate receptors (NMDARs) remains a major goal in neuropharmacology, with the potential to advance basic research and enable targeted therapies for disorders involving dysregulated glutamatergic signalling. In this volume of the Journal of General Physiology, Lotti et al. describe UCM-101, a newly optimized GluN2A-selective allosteric inhibitor derived from the weakly active scaffold TCN-213. Introduction of a single ethyl group resulted in a 7.5-fold increase in potency, yielding an inhibitor with an IC₅₀ of 110 nM at GluN1/2A receptors and up to 118-fold selectivity over other NMDAR subtypes under physiologically relevant conditions. A 1.7 Å crystal structure of the GluN1-2A ligand-binding domain (LBD) revealed that UCM-101 adopts an extended conformation spanning the inter-subunit allosteric pocket, engaging a previously unexploited \"UCM-subsite\" distinct from those used by TCN- or MPX-class modulators. Despite its novel orientation, UCM-101 stabilizes the inactive, open-clamshell conformation of the GluN1 LBD, thereby reducing glycine affinity and preventing receptor activation. Mutagenesis identified new selectivity determinants (GluN2A V529, M788, and T797) that are not utilized by TCN-201, demonstrating that different scaffolds exploit distinct microenvironments within the same allosteric site. Functionally, UCM-101 produced robust inhibition of NMDAR-mediated synaptic currents in hippocampal slices (89% at 3 μM) and displayed similar potency at triheteromeric GluN1/2A/2B receptors. Together, these findings validate the mechanistic framework for GluN2A-selective inhibition while broadening the structural landscape for ligand engagement. UCM-101 provides both a potent research tool and a promising scaffold for the development of next-generation subtype-selective NMDAR modulators.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145670581","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 critical residue mediates proper assembly and gating of GIRK2 channels. 一个关键的残基介导了GIRK2通道的适当组装和门控。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-19 DOI: 10.1085/jgp.202513817

Ha Nguyen, Jonathan Mount, Keino Hutchinson, Yihan Zhao, Yulin Zhao, Ian W Glaaser, Peng Yuan, Avner Schlessinger, Paul A Slesinger

{"title":"A critical residue mediates proper assembly and gating of GIRK2 channels.","authors":"Ha Nguyen, Jonathan Mount, Keino Hutchinson, Yihan Zhao, Yulin Zhao, Ian W Glaaser, Peng Yuan, Avner Schlessinger, Paul A Slesinger","doi":"10.1085/jgp.202513817","DOIUrl":"10.1085/jgp.202513817","url":null,"abstract":"G protein-gated inwardly rectifying potassium (GIRK) channels mediate membrane hyperpolarization in response to G protein-coupled receptor activation and are critical for regulating neuronal excitability. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for regulating the large family of inward rectifiers, and disruptions in PIP2 interactions contribute to some neurological diseases. Structural analyses have identified arginine-92 (R92) in GIRK2 as a key amino acid interacting with PIP2 as well as the potentiator cholesteryl hemisuccinate (CHS). Using electrophysiological assays and fluorescent K+ flux measurements, we show that substitutions at R92 (F, Y, or Q) disrupt PIP2 regulation, as well as G protein and alcohol activation. Cryo-EM structures of R92F and R92Q show an unexpected change in the orientation of the slide helix that leads to a \"domain swap\" between adjacent subunits in the cytoplasmic domain, producing a unique arrangement of the alcohol-binding pocket and G protein-interacting domain. These findings indicate that R92 plays a crucial role in how GIRK2 channel subunits assemble for physiological gating, and likely extend to gating of most inward rectifiers due to the high conservation of arginine in that location.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13059804/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145795470","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

Towards a unified gating scheme for the CNBD ion channel family. CNBD离子通道族的统一门控方案。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-12-11 DOI: 10.1085/jgp.202513849

Jenna L Lin, Baron Chanda

{"title":"Towards a unified gating scheme for the CNBD ion channel family.","authors":"Jenna L Lin, Baron Chanda","doi":"10.1085/jgp.202513849","DOIUrl":"10.1085/jgp.202513849","url":null,"abstract":"Cyclic nucleotide-binding domain (CNBD) channels are critical components of numerous bioelectrical processes, including cardiac pacemaking, neuronal signaling, phototransduction in the eye, and stomatal regulation in plants. While members of this channel family share a conserved overall structure, they exhibit striking differences in voltage sensitivity. Hyperpolarization-activated cyclic nucleotide-gated channels are activated by membrane hyperpolarization, whereas ether-à-go-go channels open upon depolarization. Mutagenesis and chimeragenesis studies have revealed that some mutants display bipolar gating behavior-remaining closed at intermediate membrane potentials but capable of opening in response to both hyperpolarization and depolarization. Remarkably, in certain cases, just a few mutations are sufficient to reverse the intrinsic gating polarity of the channel. This degree of diversity and plasticity in voltage-dependent gating appears to be unique to the CNBD clade and is not adequately explained by existing models. In this study, we systematically evaluate current models and propose a revised framework that better accounts for the full range of voltage-gating behaviors observed in CNBD channels.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12697245/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145727285","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

Myosin-binding protein H-like nonsense variants exhibit impaired sarcomere incorporation and alter contractility. 肌球蛋白结合蛋白h样无义变异体表现出受损的肌节结合和改变收缩性。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-11-19 DOI: 10.1085/jgp.202413630

Alejandro Alvarez-Arce, Geena E Fritzmann, Hope V Burnham, Kelly N Araujo, Alexandra Peña, Lucas M Wittenkeller, Hannah E Cizauskas, David Y Barefield

{"title":"Myosin-binding protein H-like nonsense variants exhibit impaired sarcomere incorporation and alter contractility.","authors":"Alejandro Alvarez-Arce, Geena E Fritzmann, Hope V Burnham, Kelly N Araujo, Alexandra Peña, Lucas M Wittenkeller, Hannah E Cizauskas, David Y Barefield","doi":"10.1085/jgp.202413630","DOIUrl":"10.1085/jgp.202413630","url":null,"abstract":"Heart function depends on cardiomyocyte contractile apparatus and proper sarcomere protein expression. Variants in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. Recently, a sarcomere component, myosin-binding protein-H like (MyBP-HL), was identified. MyBP-HL is mainly expressed in cardiac atria and is homologous to the last three C-terminal domains of cardiac myosin-binding protein-C (cMyBP-C). The MYBPHL R255X nonsense variant has been linked to atrial enlargement, dilated cardiomyopathy, and arrhythmias. Similar nonsense mutations in MYBPC3 are linked to hypertrophic cardiomyopathy, with these mutations preventing myofilament incorporation and the degradation of the truncated protein. However, the allele frequency of the MYBPHL R255X variant is too high in the human population to be pathogenic. We sought to determine whether MYBPHL nonsense variants impact on MyBP-HL sarcomere integration and degradation of the truncated protein, and whether the MyBPHL nonsense variants lead to changes in cardiomyocyte calcium dynamics and contractility. We mimicked human MYBPHL nonsense variants in the mouse Mybphl cDNA sequence and tested their sarcomere incorporation. We demonstrated that full-length MyBP-HL overexpression showed the expected C-zone sarcomere incorporation. Nonsense variants showed defective sarcomere incorporation. We demonstrated that full-length MyBP-HL and MyBP-HL nonsense variants were degraded by both proteasome and calpain mechanisms. We did not observe changes in calcium transients. In addition, we observed changes in contraction kinetics, including sarcomere shortening. Together, these data support the hypothesis that MYBPHL nonsense variants are functionally similar.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145551755","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

Regulation of voltage-sensing structures of CaV1.2 calcium channel by the auxiliary β3-subunit. 辅助β3-亚基对CaV1.2钙通道电压敏感结构的调控。

IF 2.9 2区医学

Journal of General Physiology Pub Date : 2026-01-05 Epub Date: 2025-11-05 DOI: 10.1085/jgp.202413739

Daniela De Giorgis, Guido Mellado, Jose Antonio Garate, Alan Neely

{"title":"Regulation of voltage-sensing structures of CaV1.2 calcium channel by the auxiliary β3-subunit.","authors":"Daniela De Giorgis, Guido Mellado, Jose Antonio Garate, Alan Neely","doi":"10.1085/jgp.202413739","DOIUrl":"10.1085/jgp.202413739","url":null,"abstract":"High voltage-activated (HVA) calcium channels (CaV) have four homologous but nonidentical repeats encompassing a voltage-sensing domain (VSD) and a quarter of the pore domain (PD). HVA can be modulated by at least two accessory subunits α2δ and CaVβ. A long-standing issue is how cytoplasmic CaVβ can shift the voltage dependence of channel opening without altering gating currents. Tracking the movement of individual VSDs by voltage-clamp fluorometry in human CaV1.2 revealed that only the VSD from the second repeat (VSD II) is perturbed by CaVβ3 in a construct combining a fluorophore-tagged VSD II (S1623C) with a quenching tryptophan within 11 Å in the PD of repeat III (E1141W). The final construct, S612C_E1141W, exhibited a biphasic voltage-dependent fluorescence whose negative phase was enhanced by CaVβ3. This behavior was well described by a kinetic model that includes three states for VSD II of which the intermediate state contributes the most to pore opening in a CaVβ-dependent manner, and that open channels with VSD II in the intermediate state would yield the lowest fluorescence emissions. Molecular dynamics simulation correlates a structure with two translocated arginines with frequent fluorophore-W contact between VSD II and the pore of open channels.","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"158 1","pages":""},"PeriodicalIF":2.9,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145446613","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