Molecular dynamics (MD) simulation of biological processes has always been a challenging task due to the long timescales of the processes involved and the large amount of output data to handle. Markov state models (MSMs) have been introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to rapidly estimate experimental properties of biological processes. Herein, we propose a method for building MSMs of ion channel permeation from MD trajectories, which directly evaluates the current flowing through the channel from the model's transition matrix (T), which is crucial for comparing simulations and experimental data.
View Article and Find Full Text PDFThis retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the numerous contributions to the field of physical chemistry in the second half of the 19th century (Nernst's equilibrium potential, based on the work of Wilhelm Ostwald, Max Planck's ion electrodiffusion, Einstein's studies of Brownian motion) which led Bernstein to propose his membrane theory in the early 1900s as an explanation of Galvani's findings and cell excitability. These processes were fully elucidated by Hodgkin and Huxley in 1952 who detailed the ionic basis of resting and action potentials, but without addressing the question of where these ions passed.
View Article and Find Full Text PDFGlioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies.
View Article and Find Full Text PDFThe malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K and Cl channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death.
View Article and Find Full Text PDFCalcium ions (Ca) enter mitochondria via the mitochondrial Ca uniporter, driven by electrical and concentration gradients. In this regard, transgenic mouse models, such as calsequestrin knockout (CSQ-KO) mice, with higher mitochondrial Ca concentrations ([Ca]), should display higher cytosolic Ca concentrations ([Ca]). However, repeated measurements of [Ca] in quiescent CSQ-KO fibers never showed a difference between WT and CSQ-KO.
View Article and Find Full Text PDFGlioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K and Cl . However, while the Cl channels participating in cell volume regulation have been clearly identified, the precise nature of the K channels involved is still uncertain.
View Article and Find Full Text PDFAll cells are capable of secreting extracellular vesicles (EVs), which are not a means to eliminate unneeded cellular compounds but represent a process to exchange material (nucleic acids, lipids and proteins) between different cells. This also happens in the brain, where EVs permit the crosstalk between neuronal and non-neuronal cells, functional to homeostatic processes or cellular responses to pathological stimuli. In brain tumors, EVs are responsible for the bidirectional crosstalk between glioblastoma cells and healthy cells, and among them, astrocytes, that assume a pro-tumoral or antitumoral role depending on the stage of the tumor progression.
View Article and Find Full Text PDFCalcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter.
View Article and Find Full Text PDFAstrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown.
View Article and Find Full Text PDFDuchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized by progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 sensors of luminal Ca2+ within the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma, is proposed to contribute to Ca2+-mediated muscle damage in DMD. To directly determine the impact of Orai1-dependent SOCE on the dystrophic phenotype, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO mice).
View Article and Find Full Text PDFRegulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl current (I ), which can be modulated by cytoplasmic Ca . Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca -permeable channel Piezo1.
View Article and Find Full Text PDFAgeing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca supply to myofibrils during excitation-contraction (EC) coupling (handled by Ca release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice.
View Article and Find Full Text PDFSkeletal muscle contraction relies on both high-fidelity calcium (Ca) signals and robust capacity for adenosine triphosphate (ATP) generation. Ca release units (CRUs) are highly organized junctions between the terminal cisternae of the sarcoplasmic reticulum (SR) and the transverse tubule (T-tubule). CRUs provide the structural framework for rapid elevations in myoplasmic Ca during excitation-contraction (EC) coupling, the process whereby depolarization of the T-tubule membrane triggers SR Ca release through ryanodine receptor-1 (RyR1) channels.
View Article and Find Full Text PDFTubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in and STIM1 and ORAI1 are the two main proteins mediating store-operated Ca entry (SOCE), a mechanism activated by depletion of intracellular Ca stores (e.g.
View Article and Find Full Text PDFStore-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of Ca2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca2+ entry, as well as sustained Ca2+ release and force generation during repetitive stimulation.
View Article and Find Full Text PDFExercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca entry (SOCE). Here, we report that SOCE, peak Ca transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca entry coincident with a modest decrease in total releasable Ca store content.
View Article and Find Full Text PDFMice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca release from SR during repetitive electrical stimulation or caffeine administration.
View Article and Find Full Text PDFIn fast-twitch fibers from adult mice Ca release units (CRUs, i.e. intracellular junctions of excitation-contraction coupling), and mitochondria are structurally linked to each other by small strands, named tethers.
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