Cysteine substitutions at A40 and G45 produce functional hemichannels, but G45C exhibits impaired function as a result of coordination of metal ions. (A–C) Current traces from oocytes are shown expressing WT Cx26 (A), A40C (B), and G45C (C). Oocytes were held at −40 mV throughout and were exposed to a low extracellular Ca²⁺ (0.2 mM) solution with and without 10 µM of the heavy metal chelator TPEN. Oocytes expressing G45C show little current in 0.2 mM Ca²⁺ in the absence of TPEN and a large potentiation >10-fold after addition of TPEN. A40C hemichannel currents are robust in the absence of TPEN, and a small increase occurred after addition. TPEN had no effect on WT Cx26 hemichannels, which were robust upon lowering Ca²⁺ to 0.2 mM. (D) Summary of the effects of TPEN. Each bar represents the fold change (mean ± SEM) in current after TPEN in 0.2 mM Ca²⁺ (n = 9 for WT Cx26, n = 17 for A40C, and n = 51 for G45C).