International Journal of Molecular Sciences

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21 pages, 3897 KiB

Open AccessArticle

A Quantitative Assay for Ca²⁺ Uptake through Normal and Pathological Hemichannels

by Chiara Nardin, Abraham Tettey-Matey, Viola Donati, Daniela Marazziti, Chiara Di Pietro, Chiara Peres, Marcello Raspa, Francesco Zonta, Guang Yang, Maryna Gorelik, Serena Singh, Lia Cardarelli, Sachdev S. Sidhu and Fabio Mammano

Int. J. Mol. Sci. 2022, 23(13), 7337; https://doi.org/10.3390/ijms23137337 - 30 Jun 2022

Cited by 7 | Viewed by 3096

Abstract

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs [...] Read more.

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca²⁺) uptake with a Ca²⁺-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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23 pages, 7167 KiB

Open AccessArticle

A Lack of GD3 Synthase Leads to Impaired Renal Expression of Connexins and Pannexin1 in St8sia1 Knockout Mice

by Diana Meter, Anita Racetin, Katarina Vukojević, Marta Balog, Vedrana Ivić, Milorad Zjalić, Marija Heffer and Natalija Filipović

Int. J. Mol. Sci. 2022, 23(11), 6237; https://doi.org/10.3390/ijms23116237 - 2 Jun 2022

Cited by 2 | Viewed by 2405

Abstract

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were [...] Read more.

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1 KO mice. After euthanasia, kidney tissue was harvested, embedded in paraffin wax, and processed for immunohistochemistry. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). We determined significantly lower expression of Cx37, Cx40, Cx45, and Panx1 in different parts of the kidneys of St8sia1 KO mice compared with WT. The most consistent decrease was found in the O.S. where all markers (Cx 37, 40, 45 and Panx1) were disrupted in St8si1 KO mice. In the CTX. region, we observed decrease in the expression of Cx37, Cx45, and Panx1, while reduced expression of Cx37 and Panx1 was more specific to IN.S. The results of the present study suggest that deficiency of GD3 synthase in St8sia1 KO mice leads to disruption of renal Cx expression, which is probably related to alteration of ganglioside composition. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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16 pages, 3340 KiB

Open AccessArticle

The Expression of Connexin 37, 40, 43, 45 and Pannexin 1 in the Early Human Retina and Choroid Development and Tumorigenesis

by Matea Žužul, Mirela Lozić, Natalija Filipović, Samir Čanović, Ana Didović Pavičić, Joško Petričević, Nenad Kunac, Violeta Šoljić, Mirna Saraga-Babić, Suzana Konjevoda and Katarina Vukojevic

Int. J. Mol. Sci. 2022, 23(11), 5918; https://doi.org/10.3390/ijms23115918 - 25 May 2022

Cited by 7 | Viewed by 1888

Abstract

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. [...] Read more.

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid. Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma. In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma. Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma. Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye. It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma. Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response. Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism. Additionally, Cx43 might be a potential biomarker of tumor prognosis. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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16 pages, 3161 KiB

Open AccessArticle

Effects of Drugs Formerly Proposed for COVID-19 Treatment on Connexin43 Hemichannels

by Axelle Cooreman, Anne Caufriez, Andrés Tabernilla, Raf Van Campenhout, Kaat Leroy, Prashant Kadam, Julen Sanz Serrano, Bruna dos Santos Rodrigues, Pieter Annaert and Mathieu Vinken

Int. J. Mol. Sci. 2022, 23(9), 5018; https://doi.org/10.3390/ijms23095018 - 30 Apr 2022

Cited by 2 | Viewed by 2966

Abstract

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir [...] Read more.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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17 pages, 7781 KiB

Open AccessArticle

Connexin Expression Is Altered in Liver Development of Yotari (dab1 -/-) Mice

by Vlatka Paštar, Mirela Lozić, Nela Kelam, Natalija Filipović, Branka Bernard, Yu Katsuyama and Katarina Vukojević

Int. J. Mol. Sci. 2021, 22(19), 10712; https://doi.org/10.3390/ijms221910712 - 2 Oct 2021

Cited by 8 | Viewed by 2918

Abstract

Disabled-1 (Dab1) protein is an intracellular adaptor of reelin signaling required for prenatal neuronal migration, as well as postnatal neurotransmission, memory formation and synaptic plasticity. Yotari, an autosomal recessive mutant of the mouse Dab1 gene is recognizable by its premature death, unstable [...] Read more.

Disabled-1 (Dab1) protein is an intracellular adaptor of reelin signaling required for prenatal neuronal migration, as well as postnatal neurotransmission, memory formation and synaptic plasticity. Yotari, an autosomal recessive mutant of the mouse Dab1 gene is recognizable by its premature death, unstable gait and tremor. Previous findings are mostly based on neuronal abnormalities caused by Dab1 deficiency, but the role of the reelin signaling pathway in nonneuronal tissues and organs has not been studied until recently. Hepatocytes, the most abundant cells in the liver, communicate via gap junctions (GJ) are composed of connexins. Cell communication disruption in yotari mice was examined by analyzing the expression of connexins (Cxs): Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 during liver development at 13.5 and 15.5 gestation days (E13.5 and E15.5). Analyses were performed using immunohistochemistry and fluorescent microscopy, followed by quantification of area percentage covered by positive signal. Data are expressed as a mean ± SD and analyzed by one-way ANOVA. All Cxs examined displayed a significant decrease in yotari compared to wild type (wt) individuals at E13.5. Looking at E15.5 we have similar results with exception of Cx37 showing negligible expression in wt. Channels formation triggered by pathological stimuli, as well as propensity to apoptosis, was studied by measuring the expression of Pannexin1 (Panx1) and Apoptosis-inducing factor (AIF) through developmental stages mentioned above. An increase in Panx1 expression of E15.5 yotari mice, as well as a strong jump of AIF in both phases suggesting that yotari mice are more prone to apoptosis. Our results emphasize the importance of gap junction intercellular communication (GJIC) during liver development and their possible involvement in liver pathology and diagnostics where they can serve as potential biomarkers and drug targets. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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Review

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27 pages, 1578 KiB

Open AccessReview

Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function

by Pía C. Burboa, Mariela Puebla, Pablo S. Gaete, Walter N. Durán and Mauricio A. Lillo

Int. J. Mol. Sci. 2022, 23(13), 7303; https://doi.org/10.3390/ijms23137303 - 30 Jun 2022

Cited by 5 | Viewed by 3371

Abstract

Microcirculation homeostasis depends on several channels permeable to ions and/or small molecules that facilitate the regulation of the vasomotor tone, hyperpermeability, the blood–brain barrier, and the neurovascular coupling function. Connexin (Cxs) and Pannexin (Panxs) large-pore channel proteins are implicated in several aspects of [...] Read more.

Microcirculation homeostasis depends on several channels permeable to ions and/or small molecules that facilitate the regulation of the vasomotor tone, hyperpermeability, the blood–brain barrier, and the neurovascular coupling function. Connexin (Cxs) and Pannexin (Panxs) large-pore channel proteins are implicated in several aspects of vascular physiology. The permeation of ions (i.e., Ca²⁺) and key metabolites (ATP, prostaglandins, D-serine, etc.) through Cxs (i.e., gap junction channels or hemichannels) and Panxs proteins plays a vital role in intercellular communication and maintaining vascular homeostasis. Therefore, dysregulation or genetic pathologies associated with these channels promote deleterious tissue consequences. This review provides an overview of current knowledge concerning the physiological role of these large-pore molecule channels in microcirculation (arterioles, capillaries, venules) and in the neurovascular coupling function. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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15 pages, 11406 KiB

Open AccessReview

Pannexins and Connexins: Their Relevance for Oocyte Developmental Competence

by Paweł Kordowitzki, Gabriela Sokołowska, Marta Wasielak-Politowska, Agnieszka Skowronska and Mariusz T. Skowronski

Int. J. Mol. Sci. 2021, 22(11), 5918; https://doi.org/10.3390/ijms22115918 - 31 May 2021

Cited by 10 | Viewed by 3642

Abstract

The oocyte is the major determinant of embryo developmental competence in all mammalian species. Although fundamental advances have been generated in the field of reproductive medicine and assisted reproductive technologies in the past three decades, researchers and clinicians are still trying to elucidate [...] Read more.

The oocyte is the major determinant of embryo developmental competence in all mammalian species. Although fundamental advances have been generated in the field of reproductive medicine and assisted reproductive technologies in the past three decades, researchers and clinicians are still trying to elucidate molecular factors and pathways, which could be pivotal for the oocyte’s developmental competence. The cell-to-cell and cell-to-matrix communications are crucial not only for oocytes but also for multicellular organisms in general. This latter mentioned communication is among others possibly due to the Connexin and Pannexin families of large-pore forming channels. Pannexins belong to a protein group of ATP-release channels, therefore of high importance for the oocyte due to its requirements of high energy supply. An increasing body of studies on Pannexins provided evidence that these channels not only play a role during physiological processes of an oocyte but also during pathological circumstances which could lead to the development of diseases or infertility. Connexins are proteins that form membrane channels and gap-junctions, and more precisely, these proteins enable the exchange of some ions and molecules, and therefore they do play a fundamental role in the communication between the oocyte and accompanying cells. Herein, the role of Pannexins and Connexins for the processes of oogenesis, folliculogenesis, oocyte maturation and fertilization will be discussed and, at the end of this review, Pannexin and Connexin related pathologies and their impact on the developmental competence of oocytes will be provided. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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13 pages, 1508 KiB

Open AccessReview

Lymphatic Connexins and Pannexins in Health and Disease

by Avigail Ehrlich, Filippo Molica, Aurélie Hautefort and Brenda R. Kwak

Int. J. Mol. Sci. 2021, 22(11), 5734; https://doi.org/10.3390/ijms22115734 - 27 May 2021

Cited by 7 | Viewed by 4533

Abstract

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these [...] Read more.

This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system. Full article

(This article belongs to the Special Issue Connexin and Pannexin Signaling in Health and Disease 2.0)

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