Journal of General Physiology最新文献

The epithelial Na channel (ENaC) is a heterotrimer whose trafficking to the apical membrane is stimulated by aldosterone. Trafficking is associated with proteolytic cleavage of the α and γ subunits. We examined the kinetics of this process to ascertain whether the observed changes could contribute to the most rapid anti-natriuretic effects (within 1-3 h) of hormone administration in rats. Infusion of aldosterone increased the abundance of cleaved αENaC and γENaC with time constants of 2.2 and 2.3 h, respectively. Decreases in full-length γENaC and increases in full-length αENaC occurred more slowly, with time constants of 22 and 17 h. Decreases in aldosterone also caused rapid decreases in cleaved and slower changes in full-length forms. Kinetic modeling suggested that the major effect of aldosterone on γENaC kinetics was on the transition from a full-length, intracellular (I) to a cleaved, membrane-associated (M) population. This rate is relatively slow (0.002-0.01 h-1) compared with rates of degradation of M (∼0.4 h-1) and I (∼0.04 h-1). Short lifetimes (∼1 h) of channels at the surface were confirmed in a mouse collecting duct cell line (mCCD). Lifetimes of full-length forms of α and γENaC were also short in whole-cell extracts of mCCD cells but were much longer in the cytoplasm of mouse tubule suspensions (10-20 h). We conclude that one effect of aldosterone in the kidney is to increase forward trafficking of ENaC to the apical membrane, where rapid degradation from the surface permits fast regulation of apical channel abundance.

Voltage-sensing domains (VSDs) are highly conserved protein modules that regulate the activation of voltage-gated ion channels. In response to membrane depolarization, positive gating charges in the S4 helix of VSDs move across the membrane electric field, which is focused at the hydrophobic constriction site (HCS) in the center of the VSD. This conformational change is translated into opening of the channel gate. Transient interactions of the gating charges with negatively charged countercharges in the adjacent helices are critical for catalyzing this state transition and for determining its voltage dependence and kinetics. However, the mechanism by which the sequential interactions between the multiple gating- and countercharges regulate these properties remains poorly understood. Here, we analyze the state transitions of the first VSD of CaV1.1 using MD simulation of the channel exposed to an electric field and site-directed mutagenesis of gating and countercharges to investigate the role of their interactions in determining the gating properties of CaV1.1. Alanine substitutions of gating charges differentially altered the kinetics or voltage dependence of activation, depending on whether they pass the HCS (R2 and R3) or not (K0, R1, and R4). Alanine substitutions of countercharges differentially altered kinetics and voltage dependence, depending on whether they facilitate the transfer of gating charges across the HCS (E100 and D126), and whether they stabilize the activated (E87, E90, and E140) or the resting state (E100, D126). Thus, our results reveal basic mechanistic principles by which variable interactions between gating charges and countercharges regulate the gating properties of voltage-gated calcium channels.

The function of the heart depends critically on the precise timing and coordination of electrical signals generated by ion channels in cardiac cells. The voltage-gated sodium current (INa) plays a pivotal role in initiating the rapid depolarization that drives each heartbeat. Two important descriptive properties of cardiac INa are its activation and inactivation midpoints, which describe the membrane voltages at which there is a 50% probability of the channel being open or unavailable, respectively. These midpoints determine the voltage range over which sodium channels contribute to the action potential and influence how easily the heart can initiate and propagate electrical signals. Because even small shifts in these kinetic parameters can affect excitability, conduction, and arrhythmia risk, they are commonly used to characterize the effects of drugs, mutations, and disease states.

Electrically active cells like cardiomyocytes show variability in their size, shape, and electrical activity. But should we expect variability in the properties of their ionic currents? In this meta-analysis, we gather and visualize measurements of two important electrophysiological parameters: the midpoints of activation and inactivation of the cardiac fast sodium current, INa. We find a considerable variation in reported mean values between experiments, with a smaller cell-to-cell variation within experiments. We show how the between-experiment variability can be decomposed into a correlated component, affecting both midpoints almost equally, and an uncorrelated component, affecting the midpoints independently, and we find that the correlated component is much larger than the uncorrelated one. We then review biological and methodological issues that might explain the observed variability and attempt to classify each as a within-experiment or a correlated or uncorrelated between-experiment effect. Although the existence of some variability in measurements of ionic currents is well-known, we believe that this is the first work to systematically review it and that the scale of the observed variability is much larger than commonly appreciated, which has implications for modelling and machine-learning as well as experimental design, interpretation, and reporting.

The heart adapts to cardiac demand via chemical modifications of contractile myofilament proteins. Many of these modifications, such as phosphorylation, occur in proteins' intrinsically disordered regions (IDRs). These IDRs, though challenging to study, are recognized as dynamic, tunable regulators of protein function. Since cardiac dysfunction often involves altered posttranslational modifications (PTMs) in myofilament proteins, understanding how IDR changes affect protein and myofilament behavior is crucial. We hypothesized that PTMs, primarily phosphorylation, regulate ABLIM1 (a myofilament protein) by altering its IDR conformational ensemble, thereby modulating its binding to other myofilament proteins. We tested this using multiscale modeling (including molecular dynamics simulations) to predict ABLIM1's conformational ensembles pre- and postphosphorylation at sites altered in a canine model of heart failure with reduced GSK3β activity. A state-based contraction model then rationalized the physiological consequences. Our data show that local physicochemical alterations from phosphorylation in ABLIM1's IDRs significantly affect its conformational ensemble. This ensemble change subsequently influences the ability of its LIM domains to interact with titin. Furthermore, using the contraction model, we show that a reduced ability to recruit myosin heads for cross-bridge formation, resulting from the modified LIM domain/titin interactions, provides a mechanism that elucidates previous findings of diminished length-dependent activation. These findings offer critical molecular insights, reframing IDRs not merely as structural noise but as key, tunable elements that control protein interactions and ultimately impact mechanical behavior in the sarcomere. This work bridges molecular disorder and biomechanical function, providing a new perspective to understand dynamic control and dysfunction in cardiomyocyte contraction.

JGP study (Woods et al. https://doi.org/10.1085/jgp.202413679) suggests that stretch activation of fast-contracting skeletal muscle fibers might increase muscle endurance by boosting force production during fatigue.

Obscurin is a large muscle protein whose multiple functions include providing mechanical strength to the M-band and linking the sarcomere to the sarcoplasmic reticulum. Mutations in obscurin are linked to various forms of muscle diseases. This study compares cardiac function in a murine model of obscurin deletion (KO) with wild-type (WT) in vivo and ex vivo. Echocardiography showed that KO hearts had larger (+20%) end-diastolic and end-systolic volumes, reduced fractional shortening, and impaired ejection fraction, consistent with dilated cardiomyopathy. However, stroke volume and cardiac output were preserved due to increased end-diastolic volume. Morphological analyses revealed reduced sarcoplasmic reticulum volume, with preserved T-tubule network. While myofilament function was preserved in isolated myofibrils and skinned trabeculae, experiments in intact trabeculae revealed that Obscn KO hearts compared with WT displayed (1) reduced active tension at high frequencies and during resting-state contractions, (2) impaired positive inotropic and lusitropic response to β-adrenergic stimulation (isoproterenol 0.1 μM), and (3) faster mechanical restitution, suggesting reduced sarcoplasmic reticulum refractoriness. Intracellular [Ca2+]i measurements showed reduced peak systolic and increased diastolic levels in KO versus WT cardiomyocytes. Western blot experiments revealed lower SERCA and phospholamban (PLB) expression and reduced PLB phosphorylation in KO mice. While action potential parameters and conduction velocity were unchanged, β-adrenergic stimulation induced more frequent spontaneous Ca2+ waves and increased arrhythmia susceptibility in KO compared with WT. Taken together, these findings suggest that obscurin deletion, in adult mice, is linked to compensated dilated cardiomyopathy, altered E-C coupling, impaired response to inotropic agents, and increased propensity to arrhythmias.

Danicamtiv is a second-generation myotropic sarcomere activator currently in clinical trials for treating heart failure with reduced ejection fraction. Initial clinical and preclinical studies suggest that danicamtiv improves upon the major shortcoming of the first-generation myotropic sarcomere activator, omecamtiv mecarbil (OM), which overly impaired diastolic function. However, no study has directly compared the in vivo cardiac effects of danicamtiv and OM to verify these claims. These direct comparisons are essential to understand the potential benefits of one drug over the other. Therefore, this study employed carefully controlled experiments with left ventricular pressure-volume loop and echocardiographic strain analysis to compare how danicamtiv and OM alter each phase of the cardiac cycle. Our results show that for similar increases in left ventricular stroke volume, danicamtiv reduced diastolic performance and myocardial relaxation less than OM. However, danicamtiv still significantly decreased diastolic function at higher doses, like OM. Furthermore, danicamtiv and OM elicited a qualitatively similar triphasic dose-response from the left ventricle. These similarities between danicamtiv and OM in the whole heart were surprising given recent evidence showing significant differences in the drugs' molecular effects on myosin mechanics. We therefore conclude that danicamtiv likely has a wider therapeutic window than OM, but may be limited by the same trade-off between systolic and diastolic performance, driven by similar underlying mechanisms.

Voltage-gated sodium channels underpin electrical signaling in sensory neurons. Their activity is an essential element in the vast majority of pain conditions, making them significant drug targets. Sensory neuron sodium channels play roles not only in afferent signaling but also in a range of efferent regulatory mechanisms. Side effects through actions on other cell types and efferent signaling are thus important issues to address during analgesic drug development. As an example, the human genetic evidence for NaV1.7 as an ideal pain target contrasts with the side effects of NaV1.7 antagonists. In this review, we describe the history and progress toward the development of useful analgesic drugs and the renewed focus on NaV1.8 as a key target in pain treatment. NaV1.8 antagonists alone or in combination with other analgesics are likely to provide new opportunities for pain relief for the vast number of people (about 33% of the population) impacted by chronic pain, particularly present in aging populations.

To study the physiological and pathological mechanisms of muscle, it is crucial to store muscle samples in ways that preserve their properties. Glycerol is commonly used for storage, as it stabilizes muscle proteins, slows enzymatic activity, and minimizes degradation. However, previous studies validating glycerol storage have not examined its effects on passive properties. In this study, mouse extensor digitorum longus (EDL) muscles were stored in 50% glycerol in relaxing solution with protease inhibitors for various durations, then rehydrated in physiological solutions to assess mechanical properties. Active properties remained unchanged, but passive stress was sensitive to glycerol storage, showing a 56.5 ± 13.6% increase after 4 days, and this effect was permanent. The increase was most pronounced at sarcomere lengths, where titin's PEVK segment extension dominates. Using gelsolin, we determined whether the passive stress increase requires the thin filament, which is known to interact with titin's PEVK region. Both glycerol-stored fibers with and without thin filament extraction exhibited increased passive stress, suggesting that the underlying mechanism is intrinsic to titin. Finally, fibers treated with methylglyoxal, a reactive carbonyl and glycating agent that forms cross-links on lysine residues, showed a significant increase in passive stress in fibers stored in relaxing solution but not in glycerol. Thus, glycerol storage elevates passive stress in a titin-specific manner, likely involving lysine residues in the PEVK. Therefore, glycerol storage should be avoided when assessing passive stiffness. We further showed that, for long-term preservation, storage of rapidly frozen muscle at -80°C is a viable option.