American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry vol:287 issue:15 pages:12250-12266
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 InsP3 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. Madine Darby Canine Kidney cells expressing 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, while 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 CytC 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.