Investigation of the roles of Ca2+ and InsP3 diffusion in the coordination of Ca2+ signals between connected hepatocytes

Glycogenolytic agonists induce coordinated Ca2+ oscillations in multicellular rat hepatocyte systems as well as in the intact liver. The coordination of intercellular Ca2+ signals requires functional gap-junction coupling. The mechanisms ensuring this coordination are not precisely known. We investigated possible roles of Ca2+ or inositol 1,4,5-trisphosphate (InsP3) as a coordinating messengers for Ca2+ spiking among connected hepatocytes. Application of ionomycin or of supra-maximal concentrations of agonists show that Ca2+ does not significantly diffuse between connected hepatocytes, although gap junctions ensure the passage of small signaling molecules, as demonstrated by FRAP experiments. By contrast, coordination of Ca2+ spiking among connected hepatocytes can be favored by a rise in the level of InsP3, via the increase of agonist concentrations, or by a shift in the affinity of InsP3 receptor for InsP3. In the same line, coordination cannot be achieved if the InsP3 is rapidly metabolized by InsP3-phosphatase in one cell of the multiplet. These results demonstrate that even if small amounts of Ca2+ diffuse across gap junctions, they most probably do not play a significant role in inducing a coordinated Ca2+ signal among connected hepatocytes. By contrast, coordination of Ca2+ oscillations is fully dependent on the diffusion of InsP3 between neighboring cells.

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Amyloid-β regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013705 ◽

2020 ◽

Vol 295 (44) ◽

pp. 15097-15111

Author(s):

Mahua Maulik ◽

Lakshmy Vasan ◽

Abhishek Bose ◽

Saikat Dutta Chowdhury ◽

Neelanjana Sengupta ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Connexin 43 ◽

Cx43 Expression ◽

Chemical Chaperone ◽

Junction Protein ◽

Gap Junction Coupling ◽

Gap Junction Protein ◽

Junction Coupling ◽

And Function

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased β-amyloid (Aβ) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aβ modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aβ can alter Cx43 gap junctions. We show that Aβ25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aβ25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aβ25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aβ-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aβ25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aβ25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aβ can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aβ modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

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Gap junctions set the speed and nucleation rate of stage I retinal waves

10.1101/368019 ◽

2018 ◽

Author(s):

Kähne Malte ◽

Rüdiger Sten ◽

Kihara Alexandre ◽

Lindner Benjamin

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Nucleation Rate ◽

Ganglion Cells ◽

Intrinsic Noise ◽

Stage I ◽

Retinal Waves ◽

Gap Junction Coupling ◽

Junction Coupling ◽

Transmission Speed

AbstractSpontaneous waves in the developing retina are essential in the formation of the retinotopic mapping in the visual system. From experiments in rabbits, it is known that the earliest type of retinal waves (stage I) is nucleated spontaneously, propagates at a speed of 451±91 μm/sec and relies on gap junction coupling between ganglion cells. Because gap junctions (electrical synapses) have short integration times, it has been argued that they cannot set the low speed of stage I retinal waves. Here, we present a theoretical study of a two-dimensional neural network of the ganglion cell layer with gap junction coupling and intrinsic noise. We demonstrate that this model can explain observed nucleation rates as well as the comparatively slow propagation speed of the waves. From the interaction between two coupled neurons, we estimate the wave speed in the model network. Furthermore, using simulations of small networks of neurons (N≤260), we estimate the nucleation rate in form of an Arrhenius escape rate. These results allow for informed simulations of a realistically sized network, yielding values of the gap junction coupling and the intrinsic noise level that are in a physiologically plausible range.Author summaryRetinal waves are a prominent example of spontaneous activity that is observed in neuronal systems of many different species during development. Spatio-temporally correlated bursts travel across the retina at a few hundred μm/sec to facilitate the maturation of the underlying neuronal circuits. Even at the earliest stage, in which the network merely consists of ganglion cells coupled by electric synapses (gap junctions), it is unclear which mechanisms are responsible for wave nucleation and transmission speed. We propose a model of gap-junction coupled noisy neurons, in which waves emerge from rare stochastic fluctuations in single cells and the wave’s transmission speed is set by the latency of the burst onset in response to gap-junction currents between neighboring cells.

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Gap Junctions Are Required for NMDA Receptor–Dependent Cell Death in Developing Neurons

Journal of Neurophysiology ◽

10.1152/jn.00362.2007 ◽

2007 ◽

Vol 98 (5) ◽

pp. 2878-2886 ◽

Cited By ~ 35

Author(s):

Juan Carlos de Rivero Vaccari ◽

Roderick A. Corriveau ◽

Andrei B. Belousov

Keyword(s):

Cell Death ◽

Nmda Receptor ◽

Gap Junctions ◽

Nmda Receptors ◽

Gap Junction ◽

Regulated Cell Death ◽

Gap Junction Coupling ◽

Nmda Receptor Function ◽

Junction Coupling ◽

Developing Neurons

A number of studies have indicated an important role for N-methyl-d-aspartate (NMDA) receptors in cell survival versus cell death decisions during neuronal development, trauma, and ischemia. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all three of these conditions. However, whether neuronal gap junctions contribute to NMDA receptor–regulated cell death is not known. Here we address the role of neuronal gap junction coupling in NMDA receptor–regulated cell death in developing neurons. We report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In contrast, increasing or decreasing NMDA receptor function when gap junction coupling is low has no or greatly reduced impact on cell survival. Pharmacological inactivation of gap junctions or knockout of neuronal connexin 36 prevents the cell death caused by NMDA receptor hypofunction or hyperfunction. The results indicate the critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons. Based on these data, we propose the novel hypothesis that NMDA receptors and gap junctions work in concert to regulate neuronal survival.

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Faculty Opinions recommendation of Enhancement of ventricular gap-junction coupling by rotigaptide.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1109232.565239 ◽

2008 ◽

Author(s):

Eric Beyer

Keyword(s):

Gap Junction ◽

Gap Junction Coupling ◽

Junction Coupling

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Abstract 336: A 14-3-3 Mode-1 Binding Motif Promotes Gap Junction Internalization During Acute Cardiac Ischemia

Circulation Research ◽

10.1161/res.113.suppl_1.a336 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

James W Smyth ◽

Jose M Sanchez ◽

Samy Lamouille ◽

Ting-Ting Hong ◽

Jacob M Vogan ◽

...

Keyword(s):

Gap Junction ◽

Protein Trafficking ◽

Connexin 43 ◽

Electrical Coupling ◽

Acute Ischemia ◽

Binding Motif ◽

Wild Type ◽

Gap Junction Coupling ◽

Junction Coupling ◽

Cell Cell

During each heartbeat, robust cell-cell electrical coupling via connexin 43 (Cx43) gap junctions allows billions of individual cardiomyocytes to contract in synchrony. Cx43 turns over rapidly, and altered Cx43 trafficking during disease contributes to the arrhythmias of sudden cardiac death. The overall phosphorylation status of the Cx43 protein is known to regulate gap junction coupling, but the role of many residue specific phosphorylation events remains unknown. One such residue, Ser373, forms a mode-1 14-3-3 binding motif upon phosphorylation. Given that 14-3-3 proteins are known to regulate protein trafficking, we hypothesized a role for Cx43 Ser373 phosphorylation in regulation of Cx43 gap junction coupling. Using Langendorff-perfused mouse hearts we find robust phosphorylation of Cx43 at Ser373 and Ser368 after 30 min of no-flow ischemia. In human cell lines, a S373A mutation ablated Cx43/14-3-3 complexing and 35 S pulse-chase revealed Cx43 S373A also experiences a longer half-life than wild-type Cx43. Previous reports have implicated phosphorylation of Cx43 Ser368 in PKC mediated Cx43 internalization. We find that upon activation of PKC, the Cx43 S373A mutant undergoes lower and more transient levels of phosphorylation at Ser368 than wild-type Cx43. Consistent with these data, siRNA-mediated ablation of 14-3-3 expression results in enlargement of gap junction plaque formation at cell-cell borders. In conclusion, we propose that phosphorylation of Cx43 Ser373 results in 14-3-3 binding which promotes and maintains phosphorylation of Cx43 Ser368 and the subsequent internalization of gap junction channels. These results identify for the first time a specific role for 14-3-3 proteins in regulation of Cx43 internalization during acute ischemia and contribute to the development of therapies aimed at preserving or enhancing gap junction coupling in the heart.

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