Objectives: 

To determine the response of hemichannels composed of connexin 43 (Cx43), a major connexin expressed extensively in astrocytes, to changes in cytoplasmic calcium ([Ca²⁺]_i).

Background: 

Connexins are transmembrane proteins that form hexameric structures, also known as hemichannels. The docking of apposing hemichannels on neighboring cells results in the formation of functional gap junction (GJ) channels. Recent evidence indicates that hemichannels, before being incorporated into GJs, may function as a release pathway for gliotransmitters like ATP, glutamate and prostaglandin-E2. Although several pathological triggers have been described, nothing is known about the physiological influences that induce gliotransmitter release via hemichannels. Here, we investigated the regulation of Cx43 hemichannel responses by [Ca²⁺]_i changes.

Results: 

The work was performed on C6 and HeLa cells stably transfected with Cx43. We used ATP release and propidium iodide dye uptake as assays for hemichannel function and Cx43 gene silencing with siRNA & the peptides gap 26/27 to suppress hemichannels. Hemichannel responses were triggered when [Ca²⁺]_i changes induced by A23187 were in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. [Ca²⁺]_i-triggered responses involved the sequential activation of a signaling cascade consisting of CaM, CaMK-II, p38 MAPK, AA, ROS and NO. This cascade was also activated by stimulation at intermediate points such as calcium-independent activation of CaM with CALP1 or exogenous application of AA. CALP1-triggered reponses did not disappear with high concentrations of this agonist, indicating that the disappearance of [Ca²⁺]_i-triggered responses with high [Ca²⁺]_i was not mediated by CaM.

Conclusions: 

These data illustrate [Ca²⁺]_i activation & inactivation of hemichannel responses and suggest the possibility that gliotransmitter release via hemichannels may function as an alternative to [Ca²⁺]_i–triggered exocytosis. In addition, [Ca²⁺]_i influences hemichannels differently as compared to gap junctions composed of the same connexin protein.