Novel Transjunctional-Voltage Dependent Gating and Unitary Conductance Properties of the Heterotypic Gap Junction Channels Formed by Cx50 and Cx50 Chimera/Mutant

Abstract

Gap junctions (GJ) are intercellular channels that connecting the cytoplasm of two cells. GJ channel conductance (Gj) is regulated by transjunctional voltage (Vj-gating), which is the voltage difference between the interiors of the cell pair. Previously, to elucidate the molecular mechanisms underlying the Cx50 GJ channel Vj-gating, domain swapping (e.g. replacing the amino-terminus of Cx50 by that of Cx36, named Cx50-Cx36N) and site-directed mutagenesis (e.g. Cx50N9R), along with dual whole-cell voltage-clamp experiments were carried out in neuroblastoma cells (N2A) (Xin et al., 2010). Here we performed studies on cell pairs forming heterotypic GJ channels between Cx50 and Cx50 chimera/mutant at macroscopic, as well as single channel level. Cx50/Cx50Cx36N heterotypic channels showed asymmetrical Vj-gating properties with respect to Vj = 0. The macroscopic Gj showed substantial reduction (by 82% at Vj = 100mV) when depolarizing Cx50-expressing cell, whereas a moderate reduction (by 61% at Vj = −100mV) when hyperpolarizing Cx50-expressing cell. This observation is in agreement with previous finding that Cx50 hemichannel gates when the cell is depolarized. Single Cx50/Cx50Cx36N heterotypic channel displayed a main conductance of ∼120pS at low Vj pulses (± 20mV), but the conductance rectified with increasing Vj pulses. Cx50/Cx50N9R heterotypic GJ channel also exhibited asymmetrical Vj-gating properties at macroscopic level, and single channel recordings indicated a main conductance of ∼150pS at Vj = 20mV. These results demonstrate that the Vj-gating properties and unitary conductance of Cx50 hemichannels were evidently modified when docking with Cx50-Cx36N or Cx50N9R mutants. These novel properties of heterotypic channels provide insights into the Vj-gating mechanisms of Cx50 GJ channels.