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

Key points Gap-junction (GJ) channels provide direct intercellular communication and are twice the length of most membrane channels, yet they often have high efficiency in permeation of ions, which is reflected by large unitary channel conductance (γj). To reveal the key factors in determining the γj of a lens GJ channel, we studied connexin 50 mutant GJ channels, where a negatively or positively charged residue was introduced into a pore-lining domain. The results indicate that the pore surface electrostatic potential is a dictating factor for the connexin 50 GJ channel γj. A change in the local resistance of the channel pore associated with these mutant channels is an important factor for their voltage-dependent gating properties. The combination of dual patch clamp and homology structure modelling is a powerful approach in revealing molecular mechanisms of GJ gating and ion permeation. Abstract 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.