Access of Metal Ions and Methanethiosulfonate Reagents to the Calcium-Gated Connexin Hemichannel Pore: Implications for the Location of the Gate

Abstract

How connexin hemichannels open and close the pore in response to changes in voltage and extracellular calcium concentrations is still unknown. Previous work has shown that conformational changes at the extracellular entrance of the pore are critical for hemichannel gating by calcium and voltage. Recently, we found that negatively charged residues in this region could interact with calcium ions to produce occlusion of the pore. These residues form a ring, raising the possibility that a calcium-bound gating ring at the entrance of the pore forms a gate. To test whether such a gating ring serves as a physical gate that prevents the access of ions and small metabolites, we assessed the calcium state-dependent accessibility of Cd2+ and MTSES to a substituted cysteine in Cx26 (Cx26G45C) located below the residues that form the ring. Extracellular application of MTSES to Xenopus oocytes expressing Cx26G45C mutants modified hemichannel currents, indicating accessibility to this residue. However, the estimated reaction rate was independent of the extracellular calcium concentration (i.e., the reaction rate was the same whether the channels were open or were closed by calcium). Similarly, Cd2+ accessibility to residue G45C was nearly independent of extracellular Ca2+ concentration. These results indicate that the calcium-gating ring does not prevent access of ions or metabolites to the inner pore and that the physical gate is located deeper into the pore from, on the intracellular side of the G45C residues and the calcium-gating ring. R01GM099490/R01GM101950.