Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca2+ and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca2+ on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca2+ imaging) methods. HCs exhibit a time- and voltage-dependent current (I hc), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca2+]o from 20 nM to 2 μM reversibly decreases I hc, decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca2+]o modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I hc and modifies its kinetics. Rapid lowering of [Ca2+]o from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I hc that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca2+]i. A step increase of [Ca2+]i evoked by photorelease of Ca2+ early during a depolarizing pulse led to a transient decrease of I hc superimposed on a growing outward I hc; a step decrease of [Ca2+]i elicited by photoactivation of a Ca2+ scavenger provoked a transient increase in I hc. Hence, it is tempting to assume that Ca2+ exerts a direct effect on Cx45 hemichannels.