Connexin mimetic peptides inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+ elevation

Connexin mimetic peptides (CxMPs), such as Gap26 and Gap27, are known as inhibitors of gap junction channels but evidence is accruing that these peptides also inhibit unapposed/non-junctional hemichannels (HCs) residing in the plasma membrane. We used voltage clamp studies to investigate the effect of Gap26/27 at the single channel level. Such an approach allows unequivocal identification of HC currents by their single channel conductance that is typically similar to 220 pS for Cx43. In HeLa cells stably transfected with Cx43 (HeLa-Cx43), Gap26/27 peptides inhibited Cx43 HC unitary currents over minutes and increased the voltage threshold for HC opening. By contrast, an elevation of intracellular calcium ([Ca2+](i)) to 200-500 nM potentiated the unitary HC current activity and lowered the voltage threshold for HC opening. Interestingly, Gap26/27 inhibited the Ca2+-potentiated HC currents and prevented lowering of the voltage threshold for HC opening. Experiments on isolated pig ventricular cardiomyocytes, which display strong endogenous Cx43 expression, demonstrated voltage-activated unitary currents with biophysical properties of Cx43 HCs that were inhibited by small interfering RNA targeting Cx43. As observed in HeLa-Cx43 cells, HC current activity in ventricular cardiomyocytes was potentiated by [Ca2+](i) elevation to 500 nM and was inhibited by Gap26/27. Our results indicate that under pathological conditions, when [Ca2+](i) is elevated, Cx43 HC opening is promoted in cardiomyocytes and CxMPs counteract this effect.