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Gap junctions, specialized membrane structures that mediate cell-to-cell communication in almost all animal tissues, are composed of channel-forming integral membrane proteins termed connexins. Most of them, particularly connexin43 (Cx43), the most ubiquitous connexin, the major connexin present in cardiac myocytes, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Some connexins, including Cx43, show mobility shifts in gel electrophoresis when cells are exposed to phosphorylating or dephosphorylating treatments. However, after exposure of rat cardiac myocytes to different uncoupling dephosphorylating agents such as H7 or butanedione monoxime, no modification in the Cx43 phosphorylation profile was generally observed. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation pattern of Cx43 or, conversely, modifications of the latter without modifications of the intercellular coupling degree, suggest that the functional state of junctional channels might rather be determined by regulatory proteins associated with Cx43. The modulation of the activity of junctional channels by protein phosphorylation/dephosphorylation processes very likely requires (as for several other membrane channels) the formation of a multiprotein complex, where pore-forming subunits bind to auxiliary proteins (e.g. scaffolding proteins, enzymes, cytoskeleton elements) that play essential roles in channel localization and activity. Such regulatory proteins, behaving as targets for phosphorylation/dephosphorylation catalysers, might in particular control the open probability of junctional channels. A schematic illustration of the regulation of Cx43-made channels by protein phosphorylation involving a partner phosphoprotein is proposed.

Gap junctional channels are essential for normal cardiac impulse propagation. In ventricular myocytes of newborn rats, channel opening requires the presence of ATP to allow protein kinase activities; otherwise, channels are rapidly deactivated by the action of endogenous protein phosphatases (PPs). The lack of influence of Mg(2+) and of selective PP2B inhibition is not in favor of the involvements of Mg(2+)-dependent PP2C and PP2B, respectively, in the loss of channel activity. Okadaic acid (1 microM) and calyculin A (100 nM), both inhibitors of PP1 and PP2A activities, significantly retarded the loss of channel activity. However, a better preservation was obtained in the presence of selective PP1 inhibitors heparin (100 microg/ml) or protein phosphatase inhibitor 2 (I2; 100 nM). Conversely, the stimulation of endogenous PP1 activity by p-nitrophenyl phosphate, in the presence of ATP, led to a progressive fading of junctional currents unless I2 was simultaneously added. Together, these results suggest that a basal phosphorylation-dephosphorylation turnover regulates gap junctional communication which is rapidly deactivated by PP1 activity when the phosphorylation pathway is hindered.

The functional state of gap junctional channels and the phosphorylation status of Connexine43 (Cx43), the major gap junctional protein in rat heart, were evaluated in primary cultures of neonatal rat cardiomyocytes. H7, able to inhibit a range of serine/threonine protein kinases, progressively reduced gap junctional conductance to approximately 13% of its initial value within 10 min except when protein phosphatase inhibitors were also present. The dephosphorylating agent 2,3-Butanedione monoxime (BDM) produced both a quick and reversible interruption of cell-to-cell communication as well as a parallel slow inhibition of junctional currents. The introduction of a non-hydrolysable ATP analogue (ATPgammaS) in the cytosol delayed the second component, suggesting that it was the consequence of protein dephosphorylation. Western blot analysis reveals 2 forms of Cx43 with different electrophoretic mobilities which correspond to its known phosphorylated and dephosphorylated forms. After exposure of the cells to H7 (1 mmol/l, 1h) or BDM (15 mmol/l, 15 min), no modification in the level of Cx43 phosphorylation was observed. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation status of Cx43 suggest that the functional state of junctional channels might rather be determined by regulatory proteins associated to Cx43.

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

Abstract: The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The junctional channels that couple the cardiomyocytes are responsible for this propagation process. These channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Four Cxs – Cx30.2, −40, −43 and −45 – have been demonstrated to be synthesized in the cardiomyocytes. In addition, each of these Cxs has a unique expression pattern in the myocardium. A fruitful approach of the role of these Cxs in the cardiac functions came with the development of transgenic mouse models. It has been shown that Cx43 was mainly involved in influx propagation in the ventricles and that inactivation in the cardiomyocytes of the gene of this Cx predisposed to development of cardiac abnormalities. Cx40 very significantly contributes to the propagation of electrical activity in the atria and the conduction system. Cx45 is essential to coordinate the synchronization of contractile activities of embryonic cardiomyocytes and for the normal progress of cardiogenesis. Finally, Cx30.2 contributes to the slowing of propagation of excitation in the atrioventricular node. These observations enable to better understand the relationships between alteration in Cx expression or gap junction remodelling and arrhythmias in the human heart. [Copyright &y& Elsevier]