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 (V(j)-gating) and the unitary gap junction channel conductance (gamma(j)). To better understand the molecular structure(s) determining the V(j)-gating properties and the gamma(j) 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 V(j) (G(j)/V(j) relationship), slowed V(j)-gating kinetics, and reduced gamma(j) as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V(j)-gating properties and gamma(j) 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 G(j)/V(j) relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (G(min)) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (gamma(j) = 132 +/- 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V(j)-gating and the gamma(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.