The Role of Amino Terminus of Mouse Cx50 in Determining Transjunctional Voltage-Dependent Gating and Unitary Conductance

摘要

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (Vj-gating) and the unitary gap junction channel conductance (γj). To better understand the molecular structure(s) determining the Vj-gating properties and the γj of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to Vj (Gj/Vj relationship), slowed Vj-gating kinetics, and reduced γj as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar Vj-gating properties and γj to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The Gj/Vj relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (Gmin) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (γj = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the Vj-gating and the γ_j, and the introduction of a positively charged Arg at the ninth position reduced the G_min with a correlated disappearance of the substate at the single channel level.