Diabetes increases the risk of developing atrial fibrillation and especially young diabetes patients have a high relative risk. Increased focus on detecting atrial fibrillation in young diabetes patients might prove beneficial, and both anticoagulation treatment and anti-arrhythmic treatment strategies should be considered as soon as possible.
Background Phosphorylation is a key regulatory event in controlling the function of the cardiac gap junction protein connexin43 (Cx43). Recently, three new phosphorylation sites (S296, S297 and S306) were identified on Cx43; two of which (S297 and S306) are dephosphorylated during ischemia. The functional significance of these new sites is currently unknown. Objective To examine the role of S296, S297 and S306 in the regulation of electrical intercellular communication. Methods To mimic constitutive dephosphorylation serine was mutated to alanine at the three sites and expressed in HeLa cells. Electrical coupling and single-channel measurements were performed by double patch clamp; protein expression levels were assayed by western blotting, localization of Cx43 and phosphorylation of S306 by immunolabeling. Free hemichannels were assessed by biotinylation. Results Macroscopic conductance in cells expressing S306A was reduced to 57 % compared to WT while coupling was not significantly changed in either S296A or S297A expressing cells. S306A expressing cells displayed similar protein and free hemichannel abundance compared to WT-Cx43, whereas the fractional area of plaques in cell to cell interfaces was increased. However, single-channel measurements showed a WT-Cx43 main state conductance of 119 pS, whereas the main state conductance of S306A channels was reduced to 95 pS. Furthermore, channel gating was affected in S306A channels. Conclusions Lack of phosphorylation at serine 306 results in reduced coupling, which can be explained by reduced single channel conductance. We suggest that dephosphorylation of S306 partly explains the electrical uncoupling seen in myocardial ischemia.
Osmotic water permeability (P(f)) was studied in spheroid-shaped human airway epithelia explants derived from nasal polyps by the use of a new improved tissue collection and isolation procedure. The fluid-filled spheroids were lined with a single cell layer with the ciliated apical cell membrane facing the outside. They were capable of surviving hours of experiment involving continuous superfusion of the bathing medium and changes of osmolarity. A new image analysis technique was developed for measuring the spheroid diameters, giving high time and measurement resolutions. The transepithelial P(f), determined by the changes of the apical solution osmolarity, was not influenced by the presence of glucose, Na(+), or Na(+)/glucose-cotransport inhibitors in the bath, but was sensitive to the aquaporin (AQP) inhibitor HgCl(2). The measured P(f) levels and the values of activation energy were in the range of those seen in AQP-associated water transport. Together, these results indicate the presence of an AQP in the apical membrane of the spheroids. Notably, identical values for P(f) were found in CF and non-CF airway preparations, as was the case also for the calculated spontaneous fluid absorption rates.
Abstract-Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing Ϸ20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH i 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart. (Circ Res. 2008;103:519-526.)Key Words: Cx43CT Ⅲ particle-receptor interaction Ⅲ gap junctions Ⅲ connexin43 Ⅲ rotigaptide C onnexins (Cxs) are integral membrane proteins that oligomerize to form intercellular channels called gap junctions (GJs). The most abundant GJ protein in a number of mammalian systems is Cx43. GJs allow passage of ions and small molecules between cells and are regulated by a variety of chemical interactions between the Cx molecule and the microenvironment. As such, GJs act as active filters to control passage of intercellular messages and modulate function.Our previous work has suggested that regulation of Cx43 results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the Cx molecule acting as a receptor for the gating particle. 1,2 Additional studies have shown that this intramolecular particle-receptor interaction can be modulated by other intermolecular interactions in the microenvironment of the GJ plaque. 3-5 Based on the particle-receptor model, we reasoned that regulation of Cx43 could be disrupted by the binding of exogenous molecules to regions of the gating particle requi...
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