Variants with increased negative electrostatic potential in the Cx50 gap junction pore increased unitary channel...

Gap junction (GJ) channels are oligomers of connexins forming channels linking neighboring cells. GJs formed by different connexins show distinct unitary channel conductance (γ_j), transjunctional voltage-dependent gating (V_j-gating) properties, and modulation by intracellular magnesium ([Mg²⁺]_i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γ_j, V_j-gating, and/or [Mg²⁺]_i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γ_j, while increasing positively charged residue (G46K) reduced the γ_j Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γ_j, V_j-gating properties, and modulation by [Mg²⁺]_i Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γ_j without a significant change in the V_j gating. In addition, elevated [Mg²⁺]_i reduced γ_j in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γ_j of this cation-preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γ_j and the [Mg²⁺]_i modulation.