title: Aspartic acid residue D3 critically determines Cx50 gap junction channel transjunctional voltage-dependent gating and unitary conductance.
abstract: 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.
authors.affiliation: Graduate Program of Neuroscience, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
authors.forename: Li
authors.initials: L
authors.lastname: Xin
authors.forename: So
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Tomitake
authors.initials: T
authors.lastname: Tsukihara
authors.forename: Donglin
authors.initials: D
authors.lastname: Bai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004553:Electric Conductivity; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021381:Protein Transport; D000099264:Connexin 50
chemical_list: D001224:Aspartic Acid; D017630:Connexins; D005136:Eye Proteins; D000099264:Connexin 50