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

Abstract

Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca(2+) concentration ([Ca(2+)]e). It is well established that hemichannel activity is inhibited at normal (∼1 mM) [Ca(2+)]e, whereas lowering [Ca(2+)]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 [Ca(2+)]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 Ca(2+) 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 (γGlu47), and is thus likely to play an important role in Ca(2+) coordination. γGlu47 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. Using a combination of quantum chemistry methods, we analyzed the interaction between γGlu47, Arg75 and Arg184 in a Cx26 hemichannel model both in the absence and in the presence of Ca(2+). We show that Ca(2+) imparts significant local structural changes and speculate that these modifications may alter the structure of the extracellular loops in Cx26, and may thus account for the mechanism of hemichannel closure in the presence of mM [Ca(2+)]e.

Keywords: Calcium ions; Charcot Marie Tooth disease; Connexin mutations; Deafness; Gating; Hybrid DFT calculations.

Graphical abstract

Fig 1

(A) Cx26 hemichannel view from the extracellular region. The two extracellular loops connecting the four transmembrane helix are not shown for clarity. Protein backbone is shown in ribbon representation, while residues mentioned in the text are shown in licorice representation: color legend: γGlu42 (green), γGlu47 (orange), Arg75 (yellow), Arg184 (red). (B) Close up view of the residues interacting with γGlu47. Part of two connexins protomers are shown in ribbons. The configuration is taken from an equilibrium MD trajectory, in which in positions 42 and 47 there were two standard (non gamma carboxylated) Glu. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

B3LYP/6-31G(d,p) optimized dyads. γGlu47-Arg75 without (A) and with (B) calcium ion, γGlu42-Arg75 without (C) and with (D) calcium ion.

Fig. 3

B3LYP/6-31G(d,p) optimized triads γGlu47-Arg75-Arg184 without (A) and with (B) calcium ion. Cluster of AA optimized at B3LYP/6-31G(d,p):PM3 level, without (C) and with (D) calcium ion; ball and stick representation is used for the high layer, licorice representation is used for the low layer. Color code for the residues in panels C and D, are the same of Fig. 1: γGlu42 (green), γGlu47 (orange), Arg75 (yellow), Arg184 (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)