Role of gamma carboxylated Glu47 in connexin 26 hemichannel regulation by extracellular Ca2+: Insight from a local quantum chemistry study

Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca2+ concentration ([Ca2+](e)). It is well established that hemichannel activity is inhibited at normal (similar to 1 mM) [Ca2+](e), whereas lowering [Ca2+](e) to micromolar levels fosters hemichannel opening. Atomic force microscopy imaging shows significant and reversible changes of pore diameter at the extracellular mouth of Cx26 hemichannels exposed to different [Ca2+](e), however, the underlying molecular mechanisms are not fully elucidated. Analysis of the crystal structure of connexin 26 (Cx26) gap junction channels, corroborated by molecular dynamics (MD) simulations, suggests that several negatively charged amino acids create a favorable environment for low-affinity Ca2+ binding within the extracellular vestibule of the Cx26 hemichannel. In particular a highly conserved glutammic acid, found in position 47 in most connexins, is thought to undergo post translational gamma carboxylation (gamma Glu47), and is thus likely to play an important role in Ca2+ coordination. gamma Glu47 may also form salt bridges with two conserved arginines (Arg75 and Arg184 in Cx26), which are considered important in stabilizing the structure of the extracellular region.