Voltage Dependent Inhibition of Cx46 Hemichannels by Calcium

Connexins (Cxs) are transmembrane proteins involved in the electrical coupling of cells, the release of signaling molecules, and cell proliferation among others. These proteins are expressed in almost every tissue of the human body and mutations in these proteins are related to several hereditary diseases. Cxs monomers oligomerize in hexamers, which traffic to the plasma membrane and form connexons or hemichannels that can act as plasma membrane channels or can travel to intercellular contact zones where they form intercellular channels known as gap junctions. The opening of the Cx hemichannels in the membrane is tightly regulated by Ca2+ and membrane voltage. This regulation prevents leakage of cellular content and its malfunction may lead to pathologic conditions. In this work we studied the reciprocal regulation of Cxs hemichannels by Ca2+ and voltage. We expressed Cx46 in Xenopus laevis oocytes, and using two electrode voltage clamp, analyzed the inhibition of currents by Ca2+. We observed that Cxs inhibition by Ca2+ is voltage dependent. The apparent Ca2+ sensitivity is increased as the membrane voltage is made more negative. This change in the affinity can be explained by a stabilization of the closed state of the Cx channel by Ca2+. We propose a model in which the calcium-bonded state prevents the opening of the hemichannel. The model allows us to determine the affinity of the closed state for calcium. In agreement with the model and suggesting a stabilization of a conformational change that narrows the pore, calcium inhibits the water flux trough Cx hemichannels. This work supports the idea that Ca2+ and voltage act synergistically to promote the closing of the pore in Cx channels.