Biology subjects

Chinthalapudi, K.

Publications and source records attributed to Chinthalapudi, K..

10 recordsLinked to original sources

bioRxiv2026

14-3-3 Enables β-Adrenergic Regulation of the Cardiac Sodium Channel at the Intercalated Disc

Background: Beta-Adrenergic stimulation enables the heart to adapt electrical activity to increased physiological demand. Although the cardiac sodium channel Nav1.5 is essential for impulse propagation, the mechanisms underlying its acute beta-adrenergic regulation, particularly within distinct cardiomyocyte membrane domains, remain poorly understood. We investigated whether the phosphorylation-sensitive adaptor protein 14-3-3 mediates this regulation. Methods and Results: Using automated patch-clamp electrophysiology in HEK293 cells and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we found that isoproterenol rapidly increased sodium current (INa) and promoted channel activation at more hyperpolarized potentials, whereas PKA inhibition reduced INa. Disruption of 14-3-3 function abolished these responses without substantially altering basal sodium current or Nav1.5 surface expression. Macropatch recordings from ventricular myocytes revealed that isoproterenol preferentially enhanced INa at the intercalated disc, an effect prevented by 14-3-3 inhibition. Proximity ligation assays further demonstrated increased Nav1.5/14-3-3 proximity following beta-adrenergic stimulation. Systematic mutagenesis identified S460 in the Nav1.5 DI-DII linker as a critical determinant of acute PKA-dependent regulation. Molecular modeling predicted that S460 phosphorylation stabilizes the Nav1.5/14-3-3 interaction. In CRISPR-engineered S460A iPSC-CMs, acute beta-adrenergic regulation of INa was abolished, whereas the isoproterenol-induced increase in conduction velocity was significantly attenuated. Computational simulations further predicted that enhanced intercalated-disc sodium current supports increased conduction velocity across varying conditions of electrical coupling and pacing rate. Conclusions: Our findings identify 14-3-3 and S460 as critical determinants of spatially selective beta-adrenergic regulation of Nav1.5 at the cardiac intercalated disc. This mechanism provides a molecular link between sympathetic signaling and cardiac sodium current regulation, supporting the concept that localized enhancement of Nav1.5 function contributes to the adaptation of cardiac conduction during increased sympathetic demand.

molecular biology↗

bioRxiv2026

Beyond Glycogen Storage: AMPKγ2 Regulates Cardiac Hypertrophy and Electrophysiology via Myosin Interaction

IntroductionVariants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. MethodsWe generated transgenic zebrafish expressing wild-type (TgWT) or pathogenic variant (TgR299Q) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. ResultsTgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations. We observed decreased AMPK phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPK{gamma}2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPK{gamma}2 localization to the myofilament. Na/Ca{superscript 2} exchanger (NCX) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPK{gamma}2-myosin interaction may promote myofilament Ca{superscript 2} retention, thereby prolonging Ca{superscript 2} transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPK{gamma}2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. ConclusionsThe PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPK{gamma}2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

physiology↗

bioRxiv2025

Structural basis of nonmuscle myosin-2 autoinhibition mechanisms

Nonmuscle myosin-2 (NM2) is a fundamental actin-based mechanochemical ATPase that regulates cellular architecture, migration, adhesion, and force generation across diverse biological contexts. NM2 function is tightly regulated by a structural transition between an autoinhibited monomeric (10S) conformation in which ATPase activity, actin binding, and filament assembly are coordinately suppressed and an enzymatically active, filamentous conformation. The autoinhibited conformation is critical for the spatial and temporal control of contractility in nonmuscle cells, yet structural insights into the 10S conformation remain largely elusive. Here, we present the cryo-EM structure of full-length human NM2B in the 10S conformation at [~]5.24 [A] resolution. The structure reveals a tri-segmented tail fold that introduces asymmetric interactions between both myosin heavy chains and associated light chains and the sequestration of key interfaces required for actin binding and filament assembly. These structural insights provide a mechanistic foundation for understanding how regulatory post-translational modifications and disease-associated mutations shift NM2 conformational equilibria and may enable the development of structure-based interventions for cytoskeletal diseases including hearing loss, neurodegeneration, and cancer.

biophysics↗

bioRxiv2025

Structural and Functional Mechanisms Underlying Activation Gate Dynamics and IFM Motif Accessibility in Human Nav1.5

Voltage-gated sodium channels are vital for regulating excitability in muscle and nerve cells, and their dysregulation is linked to a range of diseases. However, therapeutic targeting of Nav channels remains challenging due to a limited understanding of their gating mechanisms. Here, we present a cryo-EM structure of human Nav1.5 in an intermediate open state, stabilized by interactions between the N-terminal domain and the S6I segment. This structure reveals a previously uncharacterized Na+ binding site adjacent to the conserved inactivation (IFM) motif. Molecular dynamics simulations demonstrate that monovalent cations stably occupy this site, while electrophysiological recordings demonstrate that ion binding modulates IFM motif docking and fast inactivation kinetics. Our findings reveal that IFM accessibility is dynamically regulated in this intermediate state, challenging the canonical hinged-lid model of fast inactivation. Collectively, our study provides a revised structural framework for Nav1.5 gating mechanisms, suggesting an alternative pathway for ion accessibility that may inform better mechanistic and therapeutic strategies for treating Nav1.5-related cardiac arrhythmias.

biophysics↗

bioRxiv2025

Molecular Control of Non-Muscle Myosin II-A Aggregation and Intracellular Dynamics by motor- or tail-specific MYH9 Mutations

Mutations in the MYH9 gene, which encodes the actin-based molecular motor non-muscle myosin II-A (NM2-A), cause a group of blood disorders termed MYH9-related diseases (MYH9-RD). While correlation between genotype and phenotype is not well characterized at a molecular level, motor mutations seem to cause more severe phenotypes than tail mutations. Motor domain mutation N93K, previously described as activity-impairing, causes in fact an almost non-significant defect on motor function in vitro. Conversely, it increases NM2-A filament stability and interaction with the myosin chaperone UNC45a in stress fiber-forming cells. This also alters its subcellular localization and effect on adhesion dynamics. Similar cellular effects are observed in cells expressing NM2-A E1841K, a prototypical tail mutation. In cells devoid of stress fibers such as megakaryocytes, NM2-A N93K forms large, amorphous, concentration-dependent aggregates that also contain wild type NM2-A and UNC45a. Conversely, NM2-A E1841K forms concentration-independent aggregates that exclude wild type NM2-A and UNC45a. Our data shows that the N93K mutation reduces the fraction of functional cellular NM2-A by enhancing the stability of NM2-A filaments and/or promoting protein aggregation together with wild type NM2-A. Conversely, NM2-A E1841K form aggregates that do not affect wild type NM2-A. These observations are consistent with the molecular severity observed in primary cells from patients of these genotypes.

cell biology↗

bioRxiv2024

Structure and physiological investigation of arginylated actin

Actin is an evolutionarily conserved cytoskeletal protein with crucial roles in cell polarity, division, migration, and muscle contraction. Actin function is regulated in part by posttranslational modifications. One such modification in non-muscle cells is arginylation, in which an arginine residue is added to the N-terminus of {beta}-actin. What is the structure of arginylated {beta}-actin (R-{beta}-actin), are its interactions with other proteins altered and what phenotypes result when R-{beta}-actin is the sole actin isoform present in the cell? Here we report the 4.2 [A] structure of ADP-bound human R-{beta}-actin filaments, the overall structure of which is nearly identical to the filaments made of non-arginylated actin. In vitro functional assays using isoform-pure actins with defined post-translational modifications reveal that the interaction between myosin-II and actin is altered upon actin arginylation, due to frequent detachment of myosin-II from R-actin filaments. In vivo, we find that replacement of the only actin gene in Schizosaccharomyces pombe with a synthetic gene encoding R-Sp-actin reduces Arp2/3-based actin patches while thickening the formin-induced actin. Furthermore, consistent with altered interactions between myosin-II and R-actin filaments, the assembly and constriction of cytokinetic actomyosin ring are perturbed in the R-Sp-actin cells. Thus, despite the overall structural similarity of arginylated and non-arginylated actin filaments, actin arginylation affects actin filament assortment into distinct subcellular structures and its interaction with myosin II.

biochemistry↗

bioRxiv2023

Myosin 10 uses its MyTH4 and FERM domains differentially to support two aspects of spindle pole biology required for mitotic spindle bipolarity

Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10s contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10s ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.

cell biology↗

bioRxiv2022

Competition between myosin II and βH-Spectrin regulates cytoskeletal tension

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two -spectrins and two {beta}-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila {beta}-heavy Spectrin ({beta}H-Spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that {beta}H-Spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that -Spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that {beta}H-Spectrin localizes and functions independently of -Spectrin. Instead, {beta}H-Spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which {beta}H-Spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of {beta}H-Spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how {beta}H-Spectrin participates in ratcheting mechanisms associated with cell shape change.

cell biology↗

bioRxiv2022

Structural Mechanisms of Actin Isoforms

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle changes in their design principles determine the interaction with myosin motors and actin-binding proteins. The functional diversity is further increased by posttranslational modifications (PTMs). Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal -actin (3.37[A]), cardiac -actin (3.07[A]), {beta}-actin (2.99[A]), and {gamma}-actin (3.38[A]) in the Mg2+{middle dot}ADP state with their native PTMs. The structures revealed isoform-specific conformations of the N-terminus that shifts closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Retropropagated structural changes further show that myosin binding modulates actin filament structure. Further, our structures enabled us to reveal the location of disease-causing mutations and to analyze them with respect to known locations of PTMs. Collectively, the previously unknown structures of single-isotype, posttranslationally modified bare cardiac -actin, {beta}-actin, and {gamma}-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

biochemistry↗

bioRxiv2017

Mechanistic Insights Into The Active Site And Allosteric Communication Pathways In Human Nonmuscle Myosin-2C

The cyclical interaction of myosin with F-actin and nucleotides is the basis for contractility of the actin cytoskeleton. Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosins-2 isoforms. Here, we present a 2.25 [A] crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulations approaches, this study reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Genetic disruption of this pathways reduces nucleotide binding and release kinetics up to 85-fold and abolishes nonmuscle myosin-2 specific kinetic signatures. These results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

biochemistry↗