The Residues in the First Extracellular Domain Play an Important Role in Transjunctional-Voltage Dependent Gating and Unitary Channel Conductance of Cx50 Gap Junction Channels

Abstract

Gap junctions (GJ) are intercellular channels that connecting the cytoplasm of neighbor cells. GJ channels formed by Cx50 and Cx36 show drastic disparity in their unitary conductance (γj) and transjunctional voltage-dependent gating (Vj-gating), but the important underlying molecular domains/residues are not clear. Experimental evidence showed that residues in the first extracellular domain (E1) of Cx50 likely line the GJ channel pore and are important factors in determining γj and Vj-gating. We aligned the E1 sequence of Cx50 with that of Cx36 and found 10 different residues (4/10 residues involves a change in charge). We generated a chimera Cx50Cx36E1, in which the E1 of Cx50 was replaced by the E1 of Cx36, and 4 point mutations in E1 of Cx50 (where a charge change occurs, i.e. G46E, D51M, E62N and E68R). Dual patch clamp study on the homotypic GJ channels formed by the chimera or the point mutants in N2A cell pairs indicate that the Cx50Cx36E1 channel showed little change in the Vj-gating properties, but displayed a significantly reduced main single channel conductance (γj). Our studies on the point mutations of Cx50 showed that some of the mutants altered the Vj-gating properties and others changed the γj and/or the probabilities in different conducting states. Our study indicates that Cx50 E1 is an important domain in determining the Vj-gating properties and γj. Charge changes in different residues in the E1 between Cx50 and Cx36 showed different channel properties likely dictated by either their location in the pore structure or the nature of the mutation.