Putative Pore-lining Residues and Intracellular Magnesium Influence Connexin50 Unitary Gap Junction Channel Conductance
Gap junction channels (GJs) are oligomers of connexins forming channels linking neighbouring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional-voltage dependent gating (Vj-gating) properties, and magnesium modulation. We investigate here how pore-lining residues may influence these GJ properties. We mutated putative pore-lining residues in Cx50 GJ (G8E/G46E/V53E individually or in combination) to investigate γj, Vj-gating, and magnesium modulation. GJs formed by combination mutants (G8E-G46E, G46E-V53E, and G8E-G46E-V53E) were functional and showed a significantly increased γj with little change in Vj-gating, while G8E-V53E mutants failed to form functional GJs. The γjs of the mutant GJs were reduced up to 30% by inclusion of magnesium in intracellular solution. Studying the individual mutations revealed that magnesium-dependent reduction of γj are due to one or combinations of V53E and/or G46E. Increasing negatively charged glutamate residues at 53rd and/or 46th positions of Cx50 increased magnesium-sensitive ion permeation.
Gap junction channel, unitary conductance, Mg2+-dependent modulation, patch clamp, electrostatics
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