Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance

摘要

Previous studies have suggested that the aspartic acid residue (D) at the third position is critical in determining the voltage polarity of fast V j-gating of Cx50 channels. To test whether another negatively charged residue (a glutamic acid residue, E) could fulfill the role of the D3 residue, we generated the mutant Cx50D3E. V j-dependent gating properties of this mutant channel were characterized by double-patch-clamp recordings in N2A cells. Macroscopically, the D3E substitution reduced the residual conductance (G min) to near zero and outwardly shifted the half-inactivation voltage (V 0), which is a result of both a reduced aggregate gating charge (z) and a reduced free-energy difference between the open and closed states. Single Cx50D3E gap junction channels showed reduced unitary conductance (γ j) of the main open state, reduced open dwell time at ±40 mV, and absence of a long-lived substate. In contrast, a G8E substitution tested to compare the effects of the E residue at the third and eighth positions did not modify the V j-dependent gating profile or γ j. In summary, this study is the first that we know of to suggest that the D3 residue plays an essential role, in addition to serving as a negative-charge provider, as a critical determinant of the V j-dependent gating sensitivity, open-closed stability, and unitary conductance of Cx50 gap junction channels.