The Amino Terminal Domain of Connexin36 is Involved in Regulation of Gap Junction Channels by Intracellular Magnesium Ions

Abstract

Neurons in the mammalian CNS are coupled mainly by the connexin36 (Cx36) gap junction (GJ) channels, which uniquely possess a high sensitivity to intracellular Mg2+. Although a putative Mg2+ binding site was previously identified to reside in the first extracellular loop (E1) domain, the involvement of the N terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the role of the NT domain on Mg2+ modulation of Cx36 GJs. We found that charge substitutions at the 8th, 13th, and 18th positions had the most prominent effects on Mg2+ sensitivity. Notably, the A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we developed new mathematical models that took into account contingent gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance and trans junctional Mg2+ fluxes suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. All substitutions with the exception of E3Q, also exhibited reduced permeability to Mg2+, with A13K showing the largest, ~10-fold, reduction of Mg2+ permeability. Homology modeling of all examined NT variants, however, showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+. This study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.