scholarly journals Opposing modulation of Cx26 gap junctions and hemichannels by CO2

2019 ◽  
Author(s):  
Sarbjit Nijjar ◽  
Daniel Maddison ◽  
Louise Meigh ◽  
Elizabeth de Wolf ◽  
Thomas Rodgers ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Intracellular Acidification ◽  
Dye Transfer ◽  
Network Modelling ◽  
Elastic Network ◽  
Median Reduction ◽  
Whole Cell Recordings ◽  
Junction Conductance ◽  
Glucose Analogue

SummaryCx26 hemichannels open in response to moderate elevations of CO2 (PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2. Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 5.6 nS, 95% confidence interval, 3.2 to 11.9 nS) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification, but using 30 mM propionate we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 5.3 nS (2.8 to 8.3 nS). This effect of propionate was unaffected by the K125R mutation (median reduction 7.7 nS, 4.1 to 11.0 nS). pH-dependent and CO2-dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the Keratitis Ichthyosis Deafness syndrome (N14K, A40V and A88V) also abolished the CO2-dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound, is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

scholarly journals The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains.

1989 ◽  
Vol 108 (6) ◽  
pp. 2241-2254 ◽  
Author(s):  
S B Yancey ◽  
S A John ◽  
R Lal ◽  
B J Austin ◽  
J P Revel
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Communication ◽  
Amino Terminus ◽  
Dye Transfer ◽  
Amino Terminal ◽  
Antibody Binding Site ◽  
Sds Page ◽  
Junctional Permeability ◽  
And Control

Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75% of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

scholarly journals Electron microscopy and functional analysis of recombinant innexin gap junctions

2014 ◽  
Vol 70 (a1) ◽  
pp. C851-C851
Author(s):  
Atsunori Oshima ◽  
Tomohiro Matsuzawa ◽  
Kazuyoshi Murata ◽  
Kouki Nishikawa ◽  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Gap Junction ◽  
Single Particle ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Dye Transfer ◽  
Gap Junction Channels ◽  
Class Average ◽  
Detergent Micelles

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

scholarly journals Connexin 43 Expression on Peripheral Blood Eosinophils: Role of Gap Junctions in Transendothelial Migration

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Harissios Vliagoftis ◽  
Cory Ebeling ◽  
Ramses Ilarraza ◽  
Salahaddin Mahmudi-Azer ◽  
Melanie Abel ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Peripheral Blood ◽  
Connexin 43 ◽  
Transendothelial Migration ◽  
Glycyrrhetinic Acid ◽  
Dependent Manner ◽  
Dye Transfer ◽  
Blood Eosinophils

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

scholarly journals Development of an in Vitro Assay to Assess Gap Junction Activity in Cumulus-Oocyte Complexes (COC) in the Rat

2021 ◽  
Author(s):  
◽  
Shruti Patel
Keyword(s):  
Growth Factors ◽  
Gap Junctions ◽  
Gap Junction ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Dye Transfer ◽  
Antral Follicles ◽  
Fluorescence Dye

<p>The capacity of an oocyte to mature during ovarian follicular development is a key process in reproductive biology. Bidirectional communication between mammalian oocytes and their associated follicular somatic cells (cumulus-cells) is essential for oocyte maturation. Historically, studies examining the control of ovarian follicular development focused mainly on the endocrine (external) signalling but recently intraovarian (paracrine) regulation has also been shown to be important. In addition, signalling via gap junctions between follicular cells had also been crucial for oocyte maturation and follicular development. In antral follicles, gap junction activity between the oocyte and adjacent cumulus cells first increase during follicular growth and shortly before ovulation they decrease as the oocyte resumes meiosis once more before ovulation. The range of factors that modulate gap junction activity of oocyte-cumulus cell complexes (COC) is largely unknown. The aims of these studies were to develop an assay to assess the rate of transfer of low molecular weight materials from cumulus cells to the oocyte via gap junctions. The first objective was to validate a bioassay by which to test the effects of hormones, second messengers, and growth factors on gap junction activity in rat cumulus-oocyte complexes. In this study, COCs were collected from antral follicles of untreated post-pubertal Sprague Dawley rats. Gap junction activity was measured in the presence or absence of different treatments using the fluorescence dye, Calcein-AM and in the presence of a phosphodiesterase type 3 inhibitor (PDE3) milrinone. Transfer of the calcein dye from cumulus cells into the oocyte was measured at various times using CRAIC fluorescence system. The results showed that removal of the COCs from their follicular environments disrupted the gap junction activity which recovered over time in culture media. COC were sensitive to changes in pH concentration and gap junction activity could be blocked with 8 ocatnol-1 but not carbenoxolone. Treating rat COCs with dibutyryl cAMP or agents that maintained or increased intracellular cAMP levels like milrinone or forskolin were unable to modulate gap junction activity. Further, the combined effect of the oocyte-derived growth factors: growth differentiating factor 9 (GDF9) with bone morphogenetic protein 15 (BMP15) was also unable to modulate the rate of calcein dye transfer from cumulus cells to the oocyte. Ovarian steroids such as oestradiol and testosterone by themselves were unable to modulate the gap junction activity of rat COC but the combined treatment of testosterone plus forskolin or testosterone plus forskolin plus insulin-like growth factor 1 (IGF-1) increased the rate of dye transfer from cumulus cells to the oocyte. In conclusion, a fluorescence dye transfer assay was developed to measure the effects of different treatments on gap junction activity in rat COC. Under in vitro conditions, it was established that the combination of steroid and cAMP stimulators or a steroid, cAMP stimulator with IGF1 but not these reagents individually could enhance the recovery of gap junction function in rat COC. The outcomes of these experiments may help to provide new insights into developing suitable in vitro conditions, for the in vitro maturation of mammalian oocytes. Also, the newly developed assay may serve as a useful in vitro model to evaluate the effects of hormones, nutritional supplements and other factors on COC functions.</p>

scholarly journals Connexin-specific distribution within gap junctions revealed in living cells

2000 ◽  
Vol 113 (22) ◽  
pp. 4109-4120 ◽  
Author(s):  
M.M. Falk
Keyword(s):  
High Resolution ◽  
Gap Junctions ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Time Lapse ◽  
Living Cells ◽  
Dye Transfer ◽  
Channel Distribution ◽  
Specific Distribution

To study the organization of gap junctions in living cells, the connexin isotypes alpha(1)(Cx43), beta(1)(Cx32) and beta(2)(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.

A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes

1998 ◽  
Vol 111 (1) ◽  
pp. 31-43 ◽  
Author(s):  
F. Cao ◽  
R. Eckert ◽  
C. Elfgang ◽  
J.M. Nitsche ◽  
S.A. Snyder ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Lucifer Yellow ◽  
Expression System ◽  
Electrical Conductance ◽  
Dye Transfer ◽  
Specific Permeability ◽  
Fluorescent Molecules

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8–14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.

IL-1β-induced production of metalloproteinases by synovial cells depends on gap junction conductance

2002 ◽  
Vol 282 (6) ◽  
pp. C1254-C1260 ◽  
Author(s):  
Oleg V. Kolomytkin ◽  
Andrew A. Marino ◽  
David D. Waddell ◽  
J. Michael Mathis ◽  
Robert E. Wolf ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Biological Function ◽  
Synovial Cells ◽  
Gap Junction Intercellular Communication ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Junction Conductance

Synovial cells can form networks connected by gap junctions. The purpose of this study was to obtain evidence for a necessary role of gap junction intercellular communication in protein secretion by synovial cells. We developed a novel assay to measure the enzymatic activity of metalloproteinases (MMPs) produced by synovial cells in response to interleukin-1β (IL-1β) and employed the assay to explore the biological function of gap junctions. IL-1β produced a dose-dependent increase in MMP activity that was blocked by exposure to the gap junction inhibitors 18α-glycyrrhetinic acid and octanol for as few as 50 min. The inhibitors produced an immediate and marked reduction in intercellular communication, as assessed by transient current analysis using the nystatin perforated-patch method. These observations suggest that communication through gap junctions early in IL-1β signal transduction is critical to the process of cytokine-regulated secretion of MMPs by synovial cells.

scholarly journals Gap Junction Effects on Precision and Frequency of a Model Pacemaker Network

2000 ◽  
Vol 83 (2) ◽  
pp. 984-997 ◽  
Author(s):  
Katherine T. Moortgat ◽  
Theodore H. Bullock ◽  
Terrence J. Sejnowski
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Phase Distribution ◽  
Model Neuron ◽  
Spike Timing ◽  
Constant Current ◽  
Pacemaker Cells ◽  
Relay Cell ◽  
Junction Conductance

We investigated the precision of spike timing in a model of gap junction-coupled oscillatory neurons. The model incorporated the known physiology, morphology, and connectivity of the weakly electric fish's high-frequency and extremely precise pacemaker nucleus (Pn). Two neuron classes, pacemaker and relay cells, were each modeled with two compartments containing Hodgkin-Huxley sodium and potassium currents. Isolated pacemaker cells fired periodically, due to a constant current injection; relay cells were silent but slightly depolarized at rest. When coupled by gap junctions to other neurons, a model neuron, like its biological correlate, spiked at frequencies and amplitudes that were largely independent of current injections. The phase distribution in the network was labile to intracellular current injections and to gap junction conductance changes. The model predicts a biologically plausible gap junction conductance of 4–5 nS (200–250 MΩ). This results in a coupling coefficient of ∼0.02, as observed in vitro. Network parameters were varied to test which could improve the temporal precision of oscillations. Increased gap junction conductances and larger numbers of cells (holding total junctional conductance per cell constant) both substantially reduced the coefficient of variation (CV = standard deviation/mean) of relay cell spike times by 74–85% and more, and did so with lower gap junction conductance when cells were contacted axonically compared with somatically. Pacemaker cell CV was only reduced when the probability of contact was increased, and then only moderately: a fivefold increase in the probability of contact reduced CV by 35%. We conclude that gap junctions facilitate synchronization, can reduce CV, are most effective between axons, and that pacemaker cells must have low intrinsic CV to account for the low CV of cells in the biological network.

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