Biology subjects

Koval, M.

Publications and source records attributed to Koval, M..

5 recordsLinked to original sources

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2024

4-Phenylbutyric acid modulates Connexin 43 expression restricting murine-β-coronavirus infectivity and virus-induced demyelination

Gap junction intercellular communication, particularly involving Connexin 43 and Connexin 47, plays a critical role in maintaining CNS homeostasis and has been implicated in Multiple Sclerosis (MS) pathology. Thus, warranting further studies in experimental animal models to understand how modulation of Cx43 expression can influence MS pathology. Intracranial infection with murine-{beta}-coronavirus Mouse Hepatitis Virus (MHV-A59) in mice results in acute pathology characterized by high viral titers, glial activation in the brain and chronic neuroinflammatory demyelination, effectively mimicking key pathological hallmarks of MS and serving as a robust model to investigate its viral etiology. MHV-A59 infection leads to a pronounced downregulation of Cx43 during the acute phase, emphasizing its critical role in virus-induced CNS pathology. In this study, we investigated the potential of in vivo 4-phenylbutyric acid (4-PBA) administration in modulating Cx43 expression in this MHV-induced model and its consequence on chronic virus-induced demyelination. Our results reveal that 4-PBA treatment reduced acute MHV-A59 infectivity and viral spread in the brain while modulating the glial cell response, mounting host immunity. Treatment with 4-PBA effectively preserved the expression of both Cx43 and Cx47 in infected CNS cells, counteracting their infection-induced downregulation. Furthermore, MHV-A59 infection downregulated the expression of ER-resident thioredoxin family protein (ERp29), a well-known molecular chaperone of Cx43, which was rescued by 4-PBA treatment. We further validated if such downregulation of ERp29 is also evident in MS demyelinating plaques. In human MS patient-derived brain tissue, reduced Cx43 and ERp29 staining was observed in demyelinating plaques. Our studies revealed that 4-PBA treatment not only limits viral replication and spread throughout the brain but also protects the mice against severe chronic neuroinflammatory demyelination. These findings suggest that targeting Cx43 with 4-PBA holds significant therapeutic potential for addressing virus-induced neuroinflammatory demyelination and MS by preserving gap junction intercellular communication.

neuroscience↗

bioRxiv2022

Amount of Pannexin 1 in smooth muscle cells regulates sympathetic nerve induced vasoconstriction

Pannexin 1 (Panx1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in SMCs influences a-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure. To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, over-expression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Cx43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone. These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

physiology↗

bioRxiv2019

Nanotopography enhances dynamic remodeling of tight junction proteins through cytosolic complexes

The epithelial tight junction regulates barrier function and is responsive to extracellular stimuli. Here we demonstrated that contact of synthetic surfaces with defined nanotopography at the apical surface of epithelial monolayers increased paracellular permeability of macromolecules. To monitor changes in tight junction morphology in live cells, we fluorescently tagged the scaffold protein zonula occludens-1 (ZO-1) through CRISPR/Cas9-based gene editing. Contact between cells and nanostructured surfaces destabilized junction-associated ZO-1 and promoted its arrangement into highly dynamic non-junctional cytosolic complexes that averaged [~]2 m in diameter. Junction-associated ZO-1 rapidly remodeled, and we also observed the direct transformation of cytosolic complexes into junction-like structures. Claudin-family tight junction transmembrane proteins and F-actin also were associated with these ZO-1 containing cytosolic complexes. These data suggest that the cytosolic structures are novel intermediates formed in response to nanotopographic cues that facilitate rapid tight junction remodeling in order to regulate paracellular permeability.

cell biology↗

bioRxiv2019

Endothelial pannexin 1 channels control inflammation by regulating intracellular calcium

In BriefInterleukine-1 beta (IL-1{beta}) has been identified as a critical factor that contributes to the inflammatory response in cardiovascular disease (e.g., atherosclerosis). Pannexin 1 (Panx1) channel activity in endothelial cells regulates localized inflammatory cell recruitment. In response to prolonged tumor necrosis factor alpha (TNF) treatment, Yang et al. found that the Panx1 channel is targeted to the plasma membrane, where it facilitates an increase in intracellular calcium to control the production and release of cytokines including IL-1{beta}.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC=\"FIGDIR/small/750323v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@59e72borg.highwire.dtl.DTLVardef@57bd5corg.highwire.dtl.DTLVardef@50ed73org.highwire.dtl.DTLVardef@1151f56_HPS_FORMAT_FIGEXP M_FIG C_FIG AbstractThe proinflammatory cytokine IL-1{beta} is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1{beta} expression and release are tightly controlled by changes in intracellular Ca2+. In addition, purinergic signaling through ATP release has also been reported to promote IL-1{beta} production. Despite this, the mechanisms that control IL-1{beta} synthesis and expression have not been identified. The pannexin 1 (Panx1) channel has canonically been implicated in ATP release, especially during inflammation. However, resolution of purinergic signaling occurs quickly due to blood flow and the presence of ectonucleotidases. We examined Panx1 in human endothelial cells following treatment with the pro-inflammatory cytokine tumor necrosis alpha (TNF). In response to long-term TNF treatment, we identified a dramatic increase in Panx1 protein expression at the plasma membrane. Analysis by whole transcriptome sequencing (RNA-seq), qPCR, and treatment with specific kinase inhibitors, revealed that TNF signaling induced NF{kappa}{beta}-associated Panx1 transcription. Genetic inhibition of Panx1 reduced the expression and secretion of IL-1{beta}. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL1-{beta} expression was not altered after direct ATP stimulation, following degradation of ATP by apyrase, or after pharmacological blockade of P2 receptors. These data suggest that non-purinergic pathways, involving Panx1, control IL-1{beta} production. Because Panx1 forms a large pore channel, we hypothesized it may act to passively diffuse Ca2+ into the cell upon opening to regulate IL-1{beta}. High-throughput flow cytometric analysis demonstrated that TNF treatments lead to elevated intracellular Ca2+. Genetic or pharmacological inhibition of Panx1 reduced TNF-associated increases in intracellular Ca2+, and IL-1{beta} transcription. Furthermore, we found that the Ca2+-sensitive NF{kappa}{beta}-p65 protein failed to localize to the nucleus after genetic or pharmacological block of Panx1. Taken together, our study provides the first evidence that intracellular Ca2+ regulation via the Panx1 channel induces a feed-forward effect on NF{kappa}{beta} to regulate IL-1{beta} synthesis and release in endothelium during inflammation.

cell biology↗