Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Protocol for the Study of Connexin and DNA Interactions.

Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, "noncanonical" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

Diversity of Intercellular Communication Modes: A Cancer Biology Perspective.

From the moment a cell is on the path to malignant transformation, its interaction with other cells from the microenvironment becomes altered. The flow of molecular information is at the heart of the cellular and systemic fate in tumors, and various processes participate in conveying key molecular information from or to certain cancer cells. For instance, the loss of tight junction molecules is part of the signal sent to cancer cells so that they are no longer bound to the primary tumors and are thus free to travel and metastasize. Upon the targeting of a single cell by a therapeutic drug, gap junctions are able to communicate death information to by-standing cells. The discovery of the importance of novel modes of cell-cell communication such as different types of extracellular vesicles or tunneling nanotubes is changing the way scientists look at these processes. However, are they all actively involved in different contexts at the same time or are they recruited to fulfill specific tasks? What does the multiplicity of modes mean for the overall progression of the disease? Here, we extend an open invitation to think about the overall significance of these questions, rather than engage in an elusive attempt at a systematic repertory of the mechanisms at play.

Unveiling the mechanisms of trichloroethylene hypersensitivity syndrome: Exploring the role of connexin 43 gap junctions in severe skin damage.

Trichloroethylene (TCE), extensively used as an organic solvent in various industrial applications, has been identified as a causative factor in inducing hypersensitivity syndrome (THS). Currently, there is no specific treatment for THS, and most patients experience serious adverse outcomes due to extensive skin damage leading to severe infection. However, the pathogenesis of THS-associated skin damage remains unclear. This study aims to elucidate the mechanism underlying skin damage from the perspective of intercellular communication and gap junctions in THS. Our results verified that hyperactivation of connexin43 gap junctions, caused by the aberrantly elevated expression of connexin43, triggers a bystander effect that promotes apoptosis and inflammation in THS via the TNF-TNFRSF1B and mitochondria-associated pathways. Additionally, we identified the gap junction inhibitor Carbenoxolone disodium (CBX) as a promising agent for the treatment of skin damage in THS. CBX protects against inflammatory cell infiltration in the skin and decreases immune cell imbalance in the peripheral blood of THS mice. Furthermore, CBX reduces connexin43 expression, apoptosis and inflammation in THS mice. The study reveals new insights into the mechanisms underlying TCE-induced skin damage, offering a potential treatment strategy for the development of effective therapies targeting severe dermatitis induced by chemical exposure.

Assembly mechanisms of the neuronal gap junction channel connexin 36 elucidated by Cryo-EM.

Electrical synapses are essential components of neural circuits. Neuronal signal transduction across electrical synapses is primarily mediated by gap junction channels composed of Connexin36 (Cx36), the lack of which causes impaired electrical coupling between certain neurons including cortical interneurons and thalamic reticular nucleus (TRN) neurons. However, the structural basis underlying Cx36 function and assembly remains elusive. Recently, Lee et al. reported cryo-EM structures of Cx36, thus provided first insights of its gating mechanism. Here, we report a consistent cryo-EM structure of Cx36 determined in parallel, and describe unique interactions underpinning its assembly mechanism in complementary to the competing work. In particular, we found non-canonical electrostatic interactions between protomers from opposing hemichannels and a steric complementary site between adjacent protomers within a hemichannel, which together provide a structural explanation for the assembly specificity in homomeric and heteromeric gap junction channels.

Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions.

Connexins are channel-forming proteins that function to facilitate gap junctional intercellular communication. Here, we use dual cell voltage clamp and dye transfer studies to corroborate past findings showing that Cx31.1 (encoded by GJB5) is defective in gap junction channel formation, illustrating that Cx31.1 alone does not form functional gap junction channels in connexin-deficient mammalian cells. Rather Cx31.1 transiently localizes to the secretory pathway with a subpopulation reaching the cell surface, which is rarely seen in puncta reminiscent of gap junctions. Intracellular retained Cx31.1 was subject to degradation as Cx31.1 accumulated in the presence of proteasomal inhibition, had a faster turnover when Cx43 was present and ultimately reached lysosomes. Although intracellularly retained Cx31.1 was found to interact with Cx43, this interaction did not rescue its delivery to the cell surface. Conversely, the co-expression of Cx31 dramatically rescued the assembly of Cx31.1 into gap junctions where gap junction-mediated dye transfer was enhanced. Collectively, our results indicate that the localization and functional status of Cx31.1 is altered through selective interplay with co-expressed connexins, perhaps suggesting Cx31.1 is a key regulator of intercellular signaling in keratinocytes.

Simulation of gap junction formation reveals critical role of Cys disulfide redox state in connexin hemichannel docking.

Video Abstract.

A Modified Tridecapeptide Probe for Imaging Cell Junction.

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.

Capillary oxygen regulates demand-supply coupling by triggering connexin40-mediated conduction: Rethinking the metabolic hypothesis.

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+.

Fibroblast mediated dynamics in diffusively uncoupled myocytes: a simulation study using 2-cell motifs.

In healthy hearts myocytes are typically coupled to nearest neighbours through gap junctions. Under pathological conditions such as fibrosis, or in scar tissue, or across ablation lines myocytes can uncouple from their neighbours. Electrical conduction may still occur via fibroblasts that not only couple proximal myocytes but can also couple otherwise unconnected regions. We hypothesise that such coupling can alter conduction between myocytes via introduction of delays or by initiation of premature stimuli that can potentially result in reentry or conduction blocks. To test this hypothesis we have developed several 2-cell motifs and investigated the effect of fibroblast mediated electrical coupling between uncoupled myocytes. We have identified various regimes of myocyte behaviour that depend on the strength of gap-junctional conductance, connection topology, and parameters of the myocyte and fibroblast models. These motifs are useful in developing a mechanistic understanding of long-distance coupling on myocyte dynamics and enable the characterisation of interaction between different features such as myocyte and fibroblast properties, coupling strengths and pacing period. They are computationally inexpensive and allow for incorporation of spatial effects such as conduction velocity. They provide a framework for constructing scar tissue boundaries and enable linking of cellular level interactions with scar induced arrhythmia.

Adaptive remodeling of the stimulus-secretion coupling: Lessons from the 'stressed' adrenal medulla.

Stress is part of our daily lives and good health in the modern world is offset by unhealthy lifestyle factors, including the deleterious consequences of stress and associated pathologies. Repeated and/or prolonged stress may disrupt the body homeostasis and thus threatens our lives. Adaptive processes that allow the organism to adapt to new environmental conditions and maintain its homeostasis are therefore crucial. The adrenal glands are major endocrine/neuroendocrine organs involved in the adaptive response of the body facing stressful situations. Upon stress episodes and in response to activation of the sympathetic nervous system, the first adrenal cells to be activated are the neuroendocrine chromaffin cells located in the medullary tissue of the adrenal gland. By releasing catecholamines (mainly epinephrine and to a lesser extent norepinephrine), adrenal chromaffin cells actively contribute to the development of adaptive mechanisms, in particular targeting the cardiovascular system and leading to appropriate adjustments of blood pressure and heart rate, as well as energy metabolism. Specifically, this chapter covers the current knowledge as to how the adrenal medullary tissue remodels in response to stress episodes, with special attention paid to chromaffin cell stimulus-secretion coupling. Adrenal stimulus-secretion coupling encompasses various elements taking place at both the molecular/cellular and tissular levels. Here, I focus on stress-driven changes in catecholamine biosynthesis, chromaffin cell excitability, synaptic neurotransmission and gap junctional communication. These signaling pathways undergo a collective and finely-tuned remodeling, contributing to appropriate catecholamine secretion and maintenance of body homeostasis in response to stress.

Connexins 30 and 43 expression changes in relation to age-related hearing loss.

Age-related hearing loss (ARHL), also known as presbycusis, is the number one communication disorder for aging adults. Connexin proteins are essential for intercellular communication throughout the human body, including the cochlea. Mutations in connexin genes have been linked to human syndromic and nonsyndromic deafness; thus, we hypothesize that changes in connexin gene and protein expression with age are involved in the etiology of ARHL. Here, connexin gene and protein expression changes for CBA/CaJ mice at different ages were examined, and correlations were analyzed between the changes in expression levels and functional hearing measures, such as ABRs and DPOAEs. Moreover, we investigated potential treatment options for ARHL. Results showed significant downregulation of Cx30 and Cx43 gene expression and significant correlations between the degree of hearing loss and the changes in gene expression for both genes. Moreover, dose-dependent treatments utilizing cochlear cell lines showed that aldosterone hormone therapy significantly increased Cx expression. In vivo mouse treatments with aldosterone also showed protective effects on connexin expression in aging mice. Based on these functionally relevant findings, next steps can include more investigations of the mechanisms related to connexin family gap junction protein expression changes during ARHL; and expand knowledge of clinically-relevant treatment options by knowing what specific members of the Cx family and related inter-cellular proteins should be targeted therapeutically.

Estrogen-mediated coupling via gap junctions in the suprachiasmatic nucleus.

The circadian clock orchestrates many physiological and behavioural rhythms in mammals with 24-h periodicity, through a hierarchical organisation, with the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus. The circuits of the SCN generate circadian rhythms with precision, relying on intrinsic coupling mechanisms, for example, neurotransmitters like arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), neuronal gamma-aminobutyric acid (GABA) signalling and astrocytes connected by gap junctions composed of connexins (Cx). In female rodents, the presence of estrogen receptors (ERs) in the dorsal SCN suggests an influence of estrogen (E2) on the circuit timekeeping that could regulate circadian rhythm and coupling. To investigate this, we used SCN explants together with hypothalamic neurons and astrocytes. First, we showed that E2 stabilised the circadian amplitude in the SCN when rAVPs (receptor-associated vasopressin peptides) were inhibited. However, the phase delay induced by VIPAC2 (VIP receptors) inhibition remained unaffected by E2. We then showed that E2 exerted its effects in the SCN via ERß (estrogen receptor beta), resulting in increased expression of Cx36 and Cx43. Notably, specific inhibition of both connexins resulted in a significant reduction in circadian amplitude within the SCN. Remarkably, E2 restored the period with inhibited Cx36 but not with Cx43 inhibition. This implies that the network between astrocytes and neurons, responsible for coupling in the SCN, can be reinforced through E2. In conclusion, these findings provide new insights into how E2 regulates circadian rhythms ex vivo in an ERß-dependent manner, underscoring its crucial role in fortifying the SCN's rhythm.

Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury.

OBJECTIVES: To explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R. METHODS: 8-10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1ß in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH). RESULTS: Tubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R. CONCLUSIONS: The GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI. Video Abstract.