Abstract
Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, 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 Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](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 Ca(2+) entry during the rising phase of the Ca(2+) 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.
Highlights
Connexin hemichannels are Ca2ϩ-permeable plasma membrane channels that are controlled by [Ca2ϩ]i; they may contribute to Ca2ϩ oscillations
We recently reported that ATP release via hemichannels was involved in BK-induced oscillations in RBE4 brain endothelial cells [34], and we speculated that MDCK cells display a stronger ectonucleotidase activity than RBE4 cells
Because ATP release does not contribute to BK-triggered Ca2ϩ oscillations in MDCK cells, we evaluated the role of Ca2ϩ entry through hemichannels
Summary
Connexin hemichannels are Ca2ϩ-permeable plasma membrane channels that are controlled by [Ca2ϩ]i; they may contribute to Ca2ϩ oscillations. Results: Ca2ϩ oscillations triggered by bradykinin in connexin-expressing cells were inhibited by blocking hemichannel opening or by preventing their closure at high [Ca2ϩ]i; ATP-triggered oscillations were unaffected. Conclusion: Hemichannels contribute to oscillations by controlling Ca2ϩ entry. Significance: Hemichannels together with InsP3 receptors help shape agonist-induced Ca2ϩ 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 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 controlled by [Ca2؉]i. We aimed to investigate how hemichannels may contribute to Ca2؉ oscillations
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