Biology subjects

Contreras, J. E.

Publications and source records attributed to Contreras, J. E..

10 recordsLinked to original sources

bioRxiv2024

Impaired S-nitrosylation of Cx43 prevents arrhythmogenicity and myocardial injury upon cardiac stress in Duchenne Muscular Dystrophy

Connexin-43 (Cx43) plays a critical role in the propagation of action potentials and cardiac contractility. In healthy cardiomyocytes, Cx43 is mainly located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Using a mouse model of Duchenne muscular dystrophy (DMD), we previously demonstrated that Cx43 localizes to the lateral side of dystrophic cardiomyocytes, forming undocked hemichannels. {beta}-adrenergic signaling-induced cardiac stress promotes S-nitrosylation and the opening of undocked Cx43 hemichannels leading to disrupted cardiac membrane excitability and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we generated knockin DMDmdx mice with reduced levels of S-nitrosylated Cx43, by replacing cysteine 271 with a serine in one Cx43 of the unique site for S-nitrosylation of Cx43 (DMDmdx:C271S+/-). Immunofluorescence analysis revealed that cardiac Cx43 lateralization in DMDmdx:C271S+/- mice was similar to DMDmdx mice, indicating that the genetic modification did not prevent Cx43 remodeling. Upon isoproterenol treatment, DMDmdx mice displayed a higher incidence of arrhythmogenic events when compared to DMDmdx:C271S+/- mice, which more closely resemble wild-type mice. Optical mapping imaging in isolated hearts showed that DMDmdx mice displayed aberrant Ca2+ signaling and prolonged action potentials, which is restored in DMDmdx:C271S+/- mice. Isoproterenol treatment evoked severe myocardial injury in DMDmdx mice, which was significantly attenuated in DMDmdx:C271S+/- mice. Notably, DMDmdx mice treated with Gap19, a Cx43 hemichannel blocker, exhibited cardioprotection against myocardial injury. We concluded that S-nitrosylation of Cx43 proteins is a fundamental NO-mediated mechanism involved in arrhythmias and myocardial injury in DMDmdx, occurring through the opening of hemichannels following {beta}-adrenergic stress.

physiology↗

bioRxiv2024

Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using {beta}-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

physiology↗

bioRxiv2024

Large-pore connexin hemichannels function as molecule transporters independently of ion conduction

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore -- a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

biophysics↗

bioRxiv2023

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to{Delta} gra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant bubble vacuole morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, {Delta}gra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCEToxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the parasitophorous vacuole membrane (PVM), which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVMs permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47, or those with a histidine mutation, have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against Toxoplasmosis.

microbiology↗

bioRxiv2022

Microtubule-Connexin-43 regulation suppresses arrhythmias and fibrosis in Duchenne muscular dystrophy mice.

Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD) patients due to advancements in skeletal muscle therapies yet limited presence of cardiac treatments. The phosphorylation status of gap junction protein Connexin-43 (Cx43) drives Cx43 remodeling and the development of arrhythmias and fibrosis. Based on evidence that Colchicine drug treatment improves Cx43 phosphorylation and remodeling, we compared the microtubule cytoskeleton in DMD mice (mdx) versus mdx mice genetically altered to be Cx43-phosphorylation-deficient (mdxS3A). Reciprocally, we analyzed the microtubule cytoskeleton in mdx mice genetically altered to be Cx43-phospho-mimicking (mdxS3E). We found a link between the phospho-status of Connexin-43 and regulation of microtubule organization, in which phospho-dead Cx43 (S3A) inhibits improvements seen with Colchicine treatment in mdx mice, and phospho-mimic S3E promotes microtubule reorganization in mdx mice. A reduction in arrhythmias and fibrosis suggests an unsuspecting Cx43-microtubule link for translational corrective activities for DMD cardiomyopathy.

cell biology↗

bioRxiv2022

Remodeled Connexin 43 hemichannels alter cardiac excitability and promote arrhythmias

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of a dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the {beta}-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pre-treatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability and greater plasma membrane depolarization, which subsequently leads to triggered activity. These cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac stress-induced arrhythmogenicity.

physiology↗

bioRxiv2021

Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage

The connexin family is a diverse group of highly regulated non-{beta}-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3,5-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 s simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 s of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner. Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-{beta}-barrel wide-pore channels.

biophysics↗

bioRxiv2020

Connexin 43 hemichannels mediate spatial and temporal disease spread in ALS

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and may contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In human ALS tissues and biofluids, we observe that higher levels of Cx43 correlate with accelerated disease progression. Using human iPSC-derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we show that Cx43 is upregulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We then demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes, during a specific temporal window, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Our data identify Cx43 hemichannels as novel conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

neuroscience↗

bioRxiv2020

A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels.

Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small molecular dyes through these channels, we developed a protocol that renders oocytes translucent - thereby amenable to optical detection techniques - without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that molecular flux through these channels has a rate-limiting step, that the kinetic parameters of molecular transport are sensitive to modulators of channel gating and that molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, associated with molecular transport in large-pore channels.

biophysics↗

bioRxiv2019

Short-wavelength violet light (420nm) stimulates melanopsin-dependent acute alertness responses in zebrafish

Sunlight throughout the day and seasons strongly influences our biological rhythms and activity. In recent years, it has become evident that night-time overexposure to bright light in urban environments can profoundly affect physiology and behaviour in humans and animals. In particular, the artificial emission of short-wavelength light has been shown to stimulate alertness in humans, but the mechanisms remain largely unknown. Utilising a diurnal larval zebrafish model, we identified instant, non-image-forming (NIF) responses to short-wavelength violet light (~420nm), which are activated only during light exposure, and are reminiscent of alertness, including increased heart rate, enhanced locomotor activity, and pectoral fin beating (for increased oxygen supply). We further determined that these responses are driven by sympathetic neuronal circuits and depend on the zebrafish melanopsin homologue Opn4a. We also found that these responses can be modulated by the sleep-regulatory hormone melatonin, but that melatonin is not essential. Our findings reveal a previously unknown mechanism for violet light-dependent acute alertness.

animal behavior and cognition↗