RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes

Aims: Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV. Methods and results: Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity Ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of similar to 220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity Ligation studies indicated close Cx43-RyR2 apposition (