Two-color fluorescent analysis of connexin 36 turnover: relationship to functional plasticity

Wang, Helen Yanran; Lin, Ya Ping; Mitchell, Cheryl K.; Ram, Sripad; O’Brien, John

doi:10.1242/jcs.162586

Cited by 40 publications

(48 citation statements)

References 69 publications

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“…Turnover of gap junction channels was observed at electrical synapses in vivo at auditory mixed synapses on the Mauthner cell (Flores et al, ) and the existence of this phenomenon at mammalian neuronal GJs is supported by the existence of internalized annular GJs at inhibitory interneurons in the rat striatum (Fukuda, 2009). Similar turnover was observed for Cx36 in cell expression systems (Wang et al, ). This dynamic turnover must require exocytosis and endocytosis machinery and is likely strictly regulated by multiple proteins.…”

Section: What Makes An Electrical Synapse?supporting

confidence: 80%

The electrical synapse: Molecular complexities at the gap and beyond

Miller

Pereda

2017

Developmental Neurobiology

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Gap junctions underlie electrical synaptic transmission between neurons. Generally perceived as simple intercellular channels, “electrical synapses” have demonstrated to be more functionally sophisticated and structurally complex than initially anticipated. Electrical synapses represent an assembly of multiple molecules, consisting of channels, adhesion complexes, scaffolds, regulatory machinery, and trafficking proteins, all required for their proper function and plasticity. Additionally, while electrical synapses are often viewed as strictly symmetric structures, emerging evidence has shown that some components forming electrical synapses can be differentially distributed at each side of the junction. We propose that the molecular complexity and asymmetric distribution of proteins at the electrical synapse provides rich potential for functional diversity.

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Section: What Makes An Electrical Synapse?supporting

confidence: 80%

The electrical synapse: Molecular complexities at the gap and beyond

Miller

Pereda

2017

Developmental Neurobiology

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“…While Epac2 plays a significant role in E4 protection against cytokine-induced decreases in Cx36 coupling and Cx36 levels under 24 h treatment, Epac2-mediated protection was minimal under acute (1 h) treatment and no changes in Cx36 levels at the cell membrane were detected. This suggests that Epac2 regulates Cx36 coupling via slower mechanisms which occur on a time scale >1 h, such as trafficking, assembly, or turnover of Cx36 channels which occurs over ß4 h (Wang et al 2015). The robust increase in Cx36 plaque area per cell in 24 h E4 and cytokine-treated islets compared to untreated controls supports this hypothesis.…”

Section: Epac2 Mediates Camp-induced Cx36 Turnovermentioning

confidence: 81%

“…This suggests that Epac2 regulates Cx36 coupling via slower mechanisms which occur on a time scale >1 h, such as trafficking, assembly, or turnover of Cx36 channels which occurs over ∼4 h (Wang et al . ). The robust increase in Cx36 plaque area per cell in 24 h E4 and cytokine‐treated islets compared to untreated controls supports this hypothesis.…”

Section: Discussionmentioning

confidence: 97%

Exendin‐4 overcomes cytokine‐induced decreases in gap junction coupling via protein kinase A and Epac2 in mouse and human islets

Farnsworth

Walter

Piscopio

et al. 2018

The Journal of Physiology

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Key points The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing β‐cells in the islet. We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling. In both mouse and human islets exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane. Our data indicate that protein kinase A regulates β‐cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover. Increases in β‐cell coupling with exendin‐4 may protect against cytokine‐mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes. Abstract The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β‐cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β‐cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre‐diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro‐inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin‐4, which increases cAMP, can protect against cytokine‐induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin‐4 protected against cytokine‐induced decreases in coupling and preserved glucose‐stimulated calcium signalling. Exendin‐4 also protected against protein kinase Cδ‐mediated decreases in Cx36 coupling. Exendin‐4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin‐4 regulated Cx36 coupling via both protein kinase A (PKA)‐ and Epac2‐mediated mechanisms in cytokine‐treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin‐4 may protect against cytokine‐mediated β‐cell dysfunction to insulin secretion dynamics during the development of diabetes.

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“…Considering these results in conjunction with the sequences of the CT of connexins that form highly fluid GJ plaque structures (Cx26, Cx30 [ Stout et al , 2015], and Cx36 [ Wang et al , 2015]), we noticed that Cx43 differs from each of these connexins by having cytoplasmic cysteine residues within its CT. We found that a mutant form of Cx43 truncated at amino acid 268 (Cx43t268 which forms stably arranged gap junctions; Figure 1, B and C) but with an additional mutation of cysteine 260 to alanine (Cx43t268,C260A) is more mobile within GJ plaques than Cx43t268 with cysteine 260 intact (Figure 2, A and B). To test whether cysteine 260 and the other two cytoplasmic cysteine residues in the Cx43 CT act as the residues that anchor Cx43, we generated a full-length mutant form of Cx43 with all three cysteine residues in the CT mutated to alanine (Cx43C260,271,289A; referred to here as Cx43cyslCT).…”

Section: Resultsmentioning

confidence: 97%