Charge at the 46th residue of connexin 50 is crucial for the gap-junctional unitary conductance and transjunctional voltage-dependent gating.

Gap-junction (GJ) channels are twice the length of most membrane channels, yet they often have large unitary channel conductance (γj). What factors make this possibly the longest channel so efficient in passing ions are not fully clear. Here we studied the lens connexin (Cx) 50 GJs, which display one of the largest γj and the most sensitive transjunctional voltage-dependent gating (Vj gating) among all GJ channels. Introduction of charged residues into a putative pore-lining domain (the first transmembrane and the first extracellular loop border) drastically altered the apparent γj. Specifically, G46D and G46E increased the Cx50 γj from 201 to 256 and 293 pS, respectively and the G46K channel showed an apparent γj of only 20 pS. G46K also drastically altered Vj gating properties in homotypic G46K and heterotypic Cx50/G46K channels, causing an apparent loss of fast Vj-dependent gating transitions and leaving only loop gating transitions at the single channel current records. Both macroscopic and single channel currents of heterotypic Cx50/G46K channels showed a prominent rectification. Our homology structural models indicate that the pore surface electrostatic potentials are a dictating factor in determining the γj. Our data demonstrate, at the whole GJ channel level, a crucial role of the surface charge properties in the first transmembrane/first extracellular border domain in determining the efficiency of ion permeation and the Vj gating of Cx50 and possibly other GJ channels.