The N-terminal domain and voltage dependence of connexin-36 gap junction channels

Connexin-36 (Cx36) forms gap junction (GJ) channels that constitute the majority of electrical synapses in mammalian CNS and enable direct signaling between pancreatic beta cells. GJ channels are formed by the docking of two hexameric Cx hemichannels, each gating in response to the transjunctional voltage, V_j. Two distinct V_j gating mechanisms, attributed to the N-terminal domain (NT) and the first extracellular loop, are operative in each hemichannel and can modulate coupling. Uniquely among the 21 human Cx isoforms, intracellular Mg²⁺ robustly modulates Cx36 GJs, affecting the magnitude of coupling as well as sensitivity to V_j. Previously, we showed that charge substitutions E3Q, E8Q, A13K, and H18K in NT of Cx36 modified sensitivity to Mg²⁺. Here, we show that these same charge substitutions also alter V_j dependence. Mathematical modeling indicates that Mg²⁺ effects alone cannot account for the data, implicating modification of intrinsic V_j gating properties. The NT domain forms the cytoplasmic vestibule of a GJ channel and a number of residues function in sensing V_j and stabilizing open/closed configurations. Molecular dynamics simulations show that each of the NT charge substitutions altered the electrostatic profile of the channel pore and produced widespread alterations in interactions between residues in NT and the transmembrane domains that can affect the stability of the putative open conformation. Using heterotypic pairings of WT Cx36 and variants, we established a positive gating polarity for Cx36 and demonstrated polarity reversal for the E3Q substitution, properties indicative that NT-mediated gating plays a predominant role in V_j-dependence of Cx36 GJs.