Connexin 43 hemichannels mediate the Ca2+ influx induced by extracellular alkalinization

Schalper KA, Sanchez HA, Lee SC, Altenberg GA, Nathanson MH, Saez JC. Connexin 43 hemichannels mediate the Ca2+ influx induced by extracellular alkalinization. Am J Physiol Cell Physiol 299: C1504-C1515, 2010. First published September 29, 2010; doi:10.1152/ajpcell.00015.2010.-Although alkaline pH is known to trigger Ca2+ influx in diverse cells, no pH-sensitive Ca2+ channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca2+/Mg2+ concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca2+ concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La3+). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca2+ levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca2+ and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca2+ permeable, suggesting that Ca2+ influx through Cx43 HCs could account for the elevation in intracellular Ca2+ elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca2+ influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.