The rectification of heterotypic Cx46/Cx50 gap junction channels depends on intracellular magnesium

Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current-voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γ_j) rectification when 5 mmol/L Mg²⁺ was included in the patch pipette solution, while no γ_j rectification was observed when no Mg²⁺ was added. Including 5 mmol/L Mg²⁺ in pipette solution significantly decreased the γ_j of homotypic Cx46 GJ with little change in homotypic Cx50 γ_j. A missense point variant in Cx46 (E43F) reduced the Mg²⁺-dependent reduction in γ_j of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg²⁺-dependent decrease in γ_j of Cx46. A comprehensive understanding of Mg²⁺ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases.