Gap junction channels are twice the length of most membrane channels, yet their single channel currents are often large enough to be readily distinguishable under whole cell voltage clamp recording. What factors make this possibly the longest channel so efficient in passing ions are not clear. Here we used mutagenesis, dual patch clamp recording and homology modeling to study the lens Cx50 gap junction channels, which have one of the largest single channel conductance and the most sensitive transjunctional voltage-dependent gating (Vj-gating) among all characterized gap junction channels. We were able to drastically increase and decrease the unitary channel conductance (γj) of the Cx50 channel, by mutating a single pore lining residue G46, which is located at the border of the first transmembrane and the first extracellular loop domain. Specifically G46D and G46E increased the Cx50 γj from 201 to 256 and 293 pS, respectively and G46K decreased the γj to only 20 pS. The altered Vj-gating properties in homotypic and heterotypic mutant channels as well as our homology structural models provided novel insights in the possible structural factors responsible for the γj and Vj-gating. Pore surface electrostatic potentials at this domain is critical in determining the efficiency of ion permeation through this and possibly other gap junction channels.