Connexin 43 Hemichannels Contribute to Cytoplasmic Ca2+ Oscillations by Providing a Bimodal Ca2+-dependent Ca2+ Entry Pathway*

Background: Connexin hemichannels are Ca2+-permeable plasma membrane channels that are controlled by [Ca2+]i; therefore, they may contribute to Ca2+ oscillations. Results: Ca2+ oscillations triggered by bradykinin in connexin-expressing cells were inhibited by blocking hemichannel opening or by preventing their closure at high [Ca2+]i; ATP-triggered oscillations were unaffected. Conclusion: Hemichannels contribute to oscillations by controlling Ca2+ entry. Significance: Hemichannels together with InsP3 receptors help shape agonist-induced Ca2+ oscillations. Many cellular functions are driven by changes in the intracellular Ca2+ concentration ([Ca2+]i) that are highly organized in time and space. Ca2+ oscillations are particularly important in this respect and are based on positive and negative [Ca2+]i feedback on inositol 1,4,5-trisphosphate receptors (InsP3Rs). Connexin hemichannels are Ca2+-permeable plasma membrane channels that are also controlled by [Ca2+]i. We aimed to investigate how hemichannels may contribute to Ca2+ oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca2+ oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides 32Gap27/43Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca2+. Alleviating the negative feedback of [Ca2+]i on InsP3Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca2+]i but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca2+]i inhibition. Unlike interfering with the bell-shaped dependence of InsP3Rs to [Ca2+]i, CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca2+ entry during the rising phase of the Ca2+ spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.