Background: Ischemic preconditioning induces lateralization and dephosphorylation of Connexin 43 (Cx43). However, the Cx43 protein that remains at intercalated disks may be phosphorylated by casein kinase 1 (CK1) and protein kinase C (PKC), and both kinases provide cardioprotection from further ischemic injury. Here we explore the channel characteristics of a Cx43 mutant mimicking preconditioning by CK1 and PKC phosphorylation.Materials and Methods: Whole-cell patch-clamp recordings were performed in cells expressing the mutant Cx43pc (S325,328,330,368D, S365A-Cx43), and the connexin electrical behavior was analyzed at the single channel and macroscopic level.Results: Cx43pc hemichannels opened readily, whereas gap junctions channels displayed amplitudes between the wild-type and CK1 phosphorylated forms, and weaker voltage gating than either counterpart.Conclusions: Ischemic preconditioning and the ensuing phosphorylation of Cx43 by PKC may render junctional channels insensitive to transjunctional voltages, allowing the preservation of intercellular communication in ischemic conditions.

Mutations of lens connexins are linked to congenital cataracts. However, the role of connexin mutations in the development of age-related lens opacification remains largely unknown. Here, we present a focused review of the literature on lens organization and factors associated with cataract development. Several lines of evidence indicate that disturbances of the lens circulation by dysfunctional connexin channels, and/or accumulation of protein damage due to oxidative stress, are key factors in cataract development. Phosphorylation by protein kinase A improves the permeability of connexins channels to small molecules and mitigates the lens clouding induced by oxidative stress. We conclude (1) that connexin channels are central to the lens circulation and (2) that their permeability to antioxidant molecules contributes to the maintenance of lens transparency.

Connexins, elementary protein units of gap junctions, make intercellular and membrane channels that work as conduits for ions and larger molecules >1 kDa. Electrically, cells well coupled by gap junctions display a relatively uniform resting potential. In excitable tissues, gap junctions are the pathway for electrotonic propagation of action potentials. When hemichannels open in the membrane, connecting the cytoplasm and the extracellular space, the resting potential could collapse, and action potential propagation be impaired. Because connexin channels are permeated by large molecules, gap junctions are assumed to synchronize cellular functions by the sharing of second messengers, metabolites, and other substances. In turn, hemichannel opening could allow the escape of those same substances, and the uptake of extracellular molecules. Separately from their channel function, parts of the connexin molecule can induce cellular changes that suggest protein-protein interactions with the cytoskeleton, regulatory pathways, and the genomic machinery of the cells. This minireview gives an overview of connexins and discusses some of the outstanding issues in the field.

BioelectricityVol. 5, No. 3 EditorialFree AccessConnexin Connections: A Special Issue on Gap JunctionsJennifer S. Fang and Jose F. Ek-VitorinJennifer S. FangAddress correspondence to: Jennifer S. Fang, PhD, Department of Cell and Molecular Biology, School of Science and Engineering, Tulane University, New Orleans, LA 70118, USA E-mail Address: [email protected]https://orcid.org/0000-0001-5703-2239Department of Cell and Molecular Biology, School of Science and Engineering, Tulane University, New Orleans, Louisiana, USA.Search for more papers by this author and Jose F. Ek-Vitorinhttps://orcid.org/0000-0002-6487-8643Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona, USA.Search for more papers by this authorPublished Online:12 Sep 2023https://doi.org/10.1089/bioe.2023.0036AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail This year marks nearly half a century since connexin (Cx) proteins—constituent proteins of gap junction channels—were first discovered. In that time, major inroads have been made toward better understanding these understated yet enigmatic channel proteins. In this special issue, we collect several exciting primary research articles and reviews that reflect the current state of Cx and gap junction channel research.Although first imaged by electron microscopy in 1952,1 gap junctions were not named as such until 1969,2 and their constituent channel proteins were not identified for another 5 years.3 Subsequent studies in the intervening five decades have significantly expanded upon our initial understanding of Cxs and gap junctions, several questions remain.For example: Why does the mammalian genome express ∼20 different Cx isoforms, each with distinct tissue expression patterns, channel properties, and C-terminal protein domain structure? How do Cxs contribute—individually and collectively—to the physiology and the disease states of the tissues in which they are expressed? How are gap junctions innately selective for bioelectrical and organic permeants, and how is their gating regulated to properly control cell–cell transfer of these gap junction-permeable signals? What are the critical signals that pass through gap junction channels, and how does electrochemical coupling through gap junctions uniquely coordinate and control tissue development and function? Lastly, do gap junctions also possess functions beyond as intercellular conduits of electrochemical signals, and what is the contribution of this noncanonical channel-independent signaling regulation to tissue biology? These and other questions remain the subject of intense ongoing study by contemporary gap junction researchers.In this special issue, we have collected several articles, including both primary research articles and reviews, that describe the field's latest findings. Our hope is that as a special collection, these articles will help to shed new light into the important physiological roles of Cxs and gap junctions in maintaining tissue health.First, to mark the nearly half a century since Cx proteins were discovered, we reflect on how in vitro and in vivo studies have come together to reveal the importance of Cxs in our introductory review article. We describe several landmark studies that have created a comprehensive (if certainly not complete) understanding of how Cxs form gap junction channels and how these channels are regulated to mediate electrochemical cell–cell signaling.We then go on to focus on several questions of active interest among gap junction researchers, most notably recent work that has significantly expanded understanding of Cxs' function in tissue development and homeostasis. Specifically, we review site mutagenesis studies—including many conducted only within the last two decades—that have provided compelling evidence that Cxs have functions beyond their role as intercellular channels, and can also mediate a variety of noncanonical channel-independent signaling mechanisms.Separately, Dr. David Spray, a well-respected gap junction researcher, reflects on the major breakthroughs of his career. In his invited “My Experiments in Bioelectricity” feature, Dr. Spray discusses his landmark studies on regulation of Cx-specific gap junction channel voltage sensitivity. He contextualizes this work within our larger body of knowledge about the role of gap junctions in development, and further considers several key questions that remain unresolved with regard to the function of gap junction channels in isolated cells and intact tissue.It is now clear that Cxs are ubiquitously expressed in all tissues of the body and that their dysregulated expression is associated with several disease states, most notably cancer. Furthermore, individual tissues express distinct Cx expression profiles, each of which can form channels with unique channel properties. Thus, not only are Cxs critical for the health of most tissue, but they may also play diverse—and perhaps even opposing—roles within any specific tissue. In this special issue, we publish two reviews summarizing how specific Cxs regulate two distinct tissue types, one in the eye and the other in bone.In Ek-Vitorin and Jiang's review, “The role of gap junction dysfunction in the development of cataracts: from loss of cell-to-cell transfer to blurred vision,” the authors explore what is currently known about how lens Cx43, Cx46, and Cx50 contribute to fluid handling and lens transparency in the healthy eye, and how dysregulation of these Cxs (such as by abnormal phosphorylation) might lead to their dysfunction resulting in aberrant protein accumulation, lens opacity, and the development of cataracts.In their review titled “Role of Cx43 on bone cell generation, function, and survival,” Plotkin and colleagues focus on bone-expressed Cx43 and its regulation of osteoclasts, osteoblasts, and osteocytes. They explore Cx43's function as an intercellular gap junction channel in bone, where inherited mutation of Cx43 leading to the congenital disease oculodentodigital dysplasia (ODDD) has been well established. The authors then go on to discuss and integrate several new studies that describe how undocked Cx43 hemichannels also contribute to hormonal, pharmacotherapeutic, and mechanical signaling in osteoblasts and osteocytes. This review raises exciting new questions about how gap junction channel versus hemichannel activity might be regulated, and how this regulation could have potentially widespread impacts on downstream cell signaling and tissue function in health and disease.In addition to these comprehensive reviews, our special issue also includes several exciting reports of new primary research. The field has long been intrigued by how gap junction-mediated electrochemical coupling might uniquely allow for cells to function as a syncytium, and, therefore, accomplish complex information processing tasks at the tissue level far beyond the capacity of any individual cell. Blattner and Levin tackle this problem using in silico modeling of planarian tissue regeneration in their article titled “Long range communication via gap junctions as a facilitator to store morphological patterns and guide regeneration processes: A computational study.” They describe a computational model of planarian wound healing under stress based on gap junction-mediated cell–cell communication, and they show using this model that cell–cell coupling is sufficient to drive proper postinjury tissue morphogenesis and patterning. Lastly, Blattner and Levin show that simulated gap junction blockage in their computational model leads to improper bioelectrical communication and mispatterned regeneration.Finally, our special issue presents two articles focused on the upstream signals that regulate Cxs and their function, with specific focus on the widely expressed Cx isoform, Cx43. Cxs are regulated at several levels, including in their transcription, translation, junctional trafficking, and post-translational modification to control channel gating and channel-independent protein–protein interactions. In this special issue, two articles separately address transcriptional and post-translational control of Cx43, using both in vitro and in vivo models. First, Iovine and colleagues present evidence that retinoic acid (a key regulator of tissue development during embryogenesis and after injury) controls Cx43 expression in zebrafish. Using knockdown of Aldh1a2 (an enzyme responsible for synthesizing retinoic acid), the authors show that retinoic acid production is required for Cx43 upregulation and joint formation in the zebrafish fin during wound healing.Second, Ek-Vitorin et al. perform electrophysiological characterization of a putative PKC/CK-1 Cx43 phosphomimetic mutant, where they find several interesting effects on voltage sensitivity and channel gating with potential relevance to how Cx43 is phosphorylated to regulate ischemic preconditioning in the heart.Our special issue aggregates several articles that tackle numerous questions that continue to excite the gap junction field, and we collect several new findings that provide insight into these ongoing gaps in our gap junction knowledge. Furthermore, additional contributions to this special issue's focus on gap junctions are expected to be included in a follow-up collection in the issue of Bioelectricity that will be published later this year. Taken together, we hope that this special collection of gap junction research will help to bridge new scientific connections for our readers, and will encourage future study into the understated, yet endlessly fascinating, family of Cx proteins and the gap junctions that they form.References1. Robertson JD. Ultrastructure of two invertebrate synapses. Proc Soc Exp Biol Med 1953;82(2):219–223; doi: 10.3181/00379727-82-20071 Crossref, Medline, Google Scholar2. Brightman MW, Reese TS. Junctions between intimately apposed cell membranes in the vertebrate brain. J Cell Biol 1969;40(3):648–677; doi: 10.1083/jcb.40.3.648 Crossref, Medline, Google Scholar3. Goodenough DA. Bulk isolation of mouse hepatocyte gap junctions. Characterization of the principal protein, connexin. J Cell Biol 1974;61(2):557–563; doi: 10.1083/jcb.61.2.557 Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 5Issue 3Sep 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Jennifer S. Fang and Jose F. Ek-Vitorin.Connexin Connections: A Special Issue on Gap Junctions.Bioelectricity.Sep 2023.151-152.http://doi.org/10.1089/bioe.2023.0036Published in Volume: 5 Issue 3: September 12, 2023PDF download

BioelectricityVol. 4, No. 2 Call For PapersFree AccessCall for Special Issue Papers: Bioelectrical Signaling via Gap JunctionsDeadline for Manuscript Submission: March 1, 2023Guest Editors: Dr. Jose F. Ek Vitorin and Dr. Jennifer S. FangGuest Editors: Dr. Jose F. Ek VitorinThe University of Arizona, Tucson, AZ, USASearch for more papers by this author and Dr. Jennifer S. FangTulane University, New Orleans, LA, USASearch for more papers by this authorPublished Online:26 May 2022https://doi.org/10.1089/bioe.2022.29030.cfpAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Gap junctions form intercellular channels that electrochemically couple adjacent cells to spread resting and action potentials, mediate cell-cell biochemical signaling, and synchronize cell activity. In addition, undocked gap junction hemichannels can mediate depolarization and the passage of signals between cytoplasm and interstitium. Connexins – the constituent proteins of gap junctions in mammals – critically regulate a variety of complex processes during development and in disease, including cell proliferation, migration, and stem cell differentiation.This Special Issue will focus on how connexins regulate cell and tissue function by serving as cell-cell conduits of bioelectrical phenomena in healthy organ systems, as well as how these events might be altered in pathological states. Articles will explore the role of gap junctions as intercellular channels alongside growing evidence of channel-independent signal regulation, with additional consideration for how gap junctions can be novel targets for therapeutic interventions.The guest editors would welcome original research articles, reviews, perspectives, technical notes and protocols that report on recent advances in the field. Areas of interest include, but are not restricted to: Mechanisms of gap junction channel gating and permeabilityHeteromeric and heterotypic gap junction channel propertiesUpstream regulators of gap junctionsChannel-dependent and/or -independent gap junction-mediated cell signalingRole(s) of gap junctions in tissue development in normal (e.g., morphogenesis and embryogenesis) and diseased (e.g., wound healing, cancer) states.Role(s) of hemichannelsNon-mammalian gap junctions (e.g. innexins)Analogous intercellular communication in plantsContributions will receive prompt and thorough peer review. Please refer to our Instructions for Authors at www.liebertpub.com/bioe before submitting your manuscript for consideration. Also, before submitting, authors would be welcome to discuss their ideas directly with Guest Editors Dr. Jose F. Ek Vitorin (ekvitori@arizona.edu) and Dr. Jennifer S. Fang (jfang5@tulane.edu).Early submissions will receive prompt and thorough peer-review; accepted manuscripts will be published as e-pub ahead of print when-ready. This Special Issue is scheduled to appear in full online and in print in June 2023.About the JournalBioelectricity is the only peer-reviewed journal dedicated to the study of the natural electricity within living organisms and how to harness this phenomenon to treat and cure disease. The Journal publishes ground-breaking multidisciplinary research and advances documenting this next step in the evolution of how we study life. For complete tables of content and a sample issue, please visit the Bioelectricity website, www.liebertpub.com/bioe.About the PublisherMary Ann Liebert, Inc., publishers is a privately held, fully integrated media company known for establishing authoritative peer-reviewed journals in many promising areas of science and biomedical research, including Stem Cells and Development, Tissue Engineering, and The CRISPR Journal. Its biotechnology trade magazine, GEN (Genetic Engineering & Biotechnology News), was the first in its field and is today the industry's most widely read publication worldwide. A complete list of the firm's 90 journals, newsmagazines, and books is available on the Mary Ann Liebert, Inc., publishers website, www.liebertpub.com.Visit the Instructions for Authors:www.liebertpub.com/bioeSubmit your paper for peer review online:https://mc.manuscriptcentral.com/bioeFiguresReferencesRelatedDetails Volume 4Issue 2May 2022 InformationCopyright 2022, Mary Ann Liebert, Inc., publishersTo cite this article:Guest Editors: Dr. Jose F. Ek Vitorin and Dr. Jennifer S. Fang.Call for Special Issue Papers: Bioelectrical Signaling via Gap Junctions.Bioelectricity.May 2022.71-72.http://doi.org/10.1089/bioe.2022.29030.cfpPublished in Volume: 4 Issue 2: May 26, 2022PDF download

Downregulated expression of K + channels and decreased K + currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K + channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3′-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K + channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K + channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K + currents and downregulated voltage-gated K + channel 1.5 (K V 1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K + channel function and K V 1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that K V 1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca 2+ -activated K + (BK Ca ) channel currents and downregulated BK Ca channel β1 subunit (BK Ca β1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BK Ca channel activity and BK Ca β1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of K V and BK Ca channels in PASMC from patients with IPAH.

Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes’ lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels’ function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.

The synchronous heartbeat is the best and most cited example of gap junctions' electrical function. Junctional membranes represent a special case of semipermeable membrane through which the prospective separation of charges would occur between two virtually identical compartments. Junctional permeability is determined by the connexin isotype(s) composing the channels. Since not all connexins form highly permeable gap junction channels, the molecular size limit for junctional diffusion must be smaller in some tissues than in others. Stable connexin-expressing cells are obtained after transfection, using cell dilution and specific antibiotic selection of connexin-positive cell clones. An electrical rack cabinet or tower is a sturdy metallic structure to hold all electronics in a tiered order and to facilitate connections and relocation. Imperfectly chlorided or scratched wires can cause electrical noise and baseline drift. Changes on the chamber solution level that decrease the immersion of the ground wire may also cause baseline drifts.

Separate connexin domains partake in proposed gating mechanisms of gap junction channels. The amino-terminus (NT) domains, which contribute to voltage sensing, may line the channel’s cytoplasmic-facing funnel surface, stabilize the channel’s overall structure through interactions with the transmembrane domains and each other, and integrate to form a compound particle to gate the channel closed. Interactions of the carboxyl-terminus (CT) and cytoplasmic loop (CL) domains underlie voltage- and low pH-triggered channel closure. To elucidate potential cooperation of these gating mechanisms, we replaced the Cx43NT with the Cx37NT (chimera Cx43∗NT37), leaving the remainder of the Cx43 sequence, including the CT and CL, unchanged. Compared to wild-type Cx43 (Cx43WT), Cx43∗NT37 junctions exhibited several functional alterations: extreme resistance to halothane- and acidification-induced uncoupling, absence of voltage-dependent fast inactivation, longer channel open times, larger unitary channel conductances, low junctional dye permeability/permselectivity, and an overall cation selectivity more typical of Cx37WT than Cx43WT junctions. Together, these results suggest a cohesive model of channel function wherein: 1) channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the NT domains, and 2) transition between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains transduced by interactions of the pore-forming domains with either the NT, CT, or both, with the NT domains forming the gate of the completely closed channel.

The Calcium sensing receptor (CaSR) is class of G protein coupled receptors (GPCR) that can be activated by extracellular Ca 2+ and Mg 2+ . Activation of CaSR can stimulate the phospholipase C (PLC) signaling pathway that leads to synthesis of inositol 1,4,5 triphosphate (IP 3 ) and diacylglycerol (DAG). Voltage‐gated potassium (Kv) channels activity is essential for establishing resting membrane potential in a variety of cell types including pulmonary artery smooth muscle cells (PASMC). Indeed, stimulation or inhibition of K + channels can easily determine cell fate, as K + channel activity is associated with cell proliferation, survival and apoptosis. It has been reported that some K + channel classes are regulated by GPCRs. For example, Kv1.2, and members of the two‐pore domain K + channel family (TASK‐1 and TASK‐3) were shown to be inhibited by GPCR activation. Interestingly, Kv1.5 channel activity can also be suppressed by stimulation of GPCR via the Src tyrosine kinase pathway. However there are no reports to date that have shown any functional interaction between CaSR and Kv1.5. In this present study, we examined the effects of CaSR activation on Kv1.5 channel activity by transfection of KCNA5, which encodes for the Kv1.5 pore‐forming α subunit, alone or cotransfection of KCNA5/CaSR in HEK293 cells. Overexpression of KCNA5 and CaSR/KCNA5 in HEK293 cells were confirmed by Western blot and Immunofluorescence. Kv1.5 current amplitude (I Kv1.5 ) was significantly attenuated by 1.8 mM external Ca 2+ in cells transfected with both CaSR/KCNA5 but not in cells transfected with KCNA5 alone. To determine whether this Ca 2+ induced decrease of I Kv1.5 was due to activation of PLC‐DAG‐IP 3 signaling cascade we used the PLC inhibitor U‐73122. Preliminary data showed that 10uM U‐73122 was capable of antagonizing the external Ca 2+ induced decrease in I Kv1.5 . This would suggests that activation of CaSR by way of external Ca 2+ is capable of inhibiting Kv1.5 channel activity via PLC signaling pathway Support or Funding Information Research supported by NIH Grants (HL115014, HL066012 and HL098053).

Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A, C61A, C65A, C187A, C192A, C198A (designated as C(6)A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and-Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 poreforming domain able to open as a GJC.

The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore's limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

BACKGROUND: Varying strategies are currently being evaluated to develop tissue-engineered constructs for the treatment of ischemic heart disease. This study examines an angiogenic and biodegradable cardiac construct seeded with neonatal cardiomyocytes for the treatment of chronic heart failure (CHF).METHODS: We evaluated a neonatal cardiomyocyte (NCM)-seeded 3-dimensional fibroblast construct (3DFC) in vitro for the presence of functional gap junctions and the potential of the NCM-3DFC to restore left ventricular (LV) function in an in vivo rat model of CHF at 3 weeks after permanent left coronary artery ligation.RESULTS: The NCM-3DFC demonstrated extensive cell-to-cell connectivity after dye injection. At 5 days in culture, the patch contracted spontaneously in a rhythmic and directional fashion at 43 +/- 3 beats/min, with a mean displacement of 1.3 +/- 0.3 mm and contraction velocity of 0.8 +/- 0.2 mm/sec. The seeded patch could be electrically paced at nearly physiologic rates (270 +/- 30 beats/min) while maintaining coordinated, directional contractions. Three weeks after implantation, the NCM-3DFC improved LV function by increasing (p < 0.05) ejection fraction 26%, cardiac index 33%, dP/dt(+) 25%, dP/dt(-) 23%, and peak developed pressure 30%, while decreasing (p < 0.05) LV end diastolic pressure 38% and the time constant of relaxation (Tau) 16%. At 18 weeks after implantation, the NCM-3DFC improved LV function by increasing (p < 0.05) ejection fraction 54%, mean arterial pressure 20%, dP/dt(+) 16%, dP/dt(-) 34%, and peak developed pressure 39%.CONCLUSIONS: This study demonstrates that a multicellular, electromechanically organized cardiomyocyte scaffold, constructed in vitro by seeding NCM onto 3DFC, can improve LV function long-term when implanted in rats with CHF. (C) 2014 International Society for Heart and Lung Transplantation. All rights reserved.

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.

Background: While direct cell injections have been the main focus of cell-based therapies for chronic heart failure (CHF), newer approaches utilizing tissue engineered scaffolds have evolved. In CHF, we have shown that a 3-dimensional fibroblast construct (3DFC) increases myocardial blood flow and promotes angiogenesis but does not reverse maladaptive left ventricular (LV) remodeling. To attenuate remodeling requires repopulating the infarcted wall with new viable cells. We propose a neonatal cardiomyocyte seeded 3DFC (NC-3DFC) to salvage damaged myocardium and improve LV function. Methods: Cardiomyocytes are isolated from 24-48 hour old neonatal Sprague Dawley rat hearts then differentially plated in Ham's F-12 with 100mg/ml BSA, seeded onto the 3DFC, and implanted 3 weeks after left coronary artery ligation. Hemodynamic and echocardiographic studies are performed 3 weeks later. Connexin formation of NC-3DFC cultured for 6 days were evaluated using single cell dye injections. Results: The NC-3DFC contracts in a consistent rhythmic and directional fashion, spontaneously beating at 43 ± 3 bpm with a mean displacement of 1.3 ± 0.3 mm and contraction velocity of 0.8 ± 0.2 mm/sec (N = 10). Additionally, the NC-3DFC, can be electrically paced at near physiological rates, (270 ± 30 bpm). In rats with ejections fraction (EF) ≤ 35% at 3 weeks post MI, NC-3DFC implanted 3 weeks post MI increases (p < 0.05) EF from 31% ± 2 to 39% ± 1, cardiac index from 0.46 ± 0.05 to 0.61 ± 0.06 mL/(g min), and fractional shortening from 13% ± 1 to 17% ± 1, while end diastolic pressure decreases from 24 ± 2 to 14 ± 3 mmHg. Functional connexins are present at 48 hours and display increased connectivity through 6 days. Single cell dye injections show immediate intercellular dye transfer that is blocked by halothane, demonstrating that connexins are responsible for the cell-to-cell communication. Conclusion: Neonatal cardiomyocytes seeded and co-cultured onto biodegradable 3DFC allows for cellular survival, communication, and electrical coupling with resultant improvement in LV function in rats with CHF. This newly seeded NC-3DFC scaffold is a novel cell delivery system to potentially treat heart failure.

Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.