Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as Vj-gating) and display a wide range of unitary channel conductance (γj), yet the domains responsible for Vj-gating and γj are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in Vj-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct Vj-gating and γj from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γj, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in Vj-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in Vj-gating and γj. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.