The carboxyl terminal domain regulates the unitary conductance and voltagedependence of connexin40 gap junction channels

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain m odel, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa, Here, we report similar interactions in the establishment of th e unitary conductance and voltage-dependent profile of Cx40 in N2A cells. T wo mean unitary conductance values ("lower conductance" and ''main") were d etected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (C x40tr248) caused the disappearance of the lower-conductance state. Coexpres sion of Cx40tr248 with the CT fragment of either Cx40 (homodomain interacti ons) or Cx43 (heterodomain interactions) rescued the unitary conductance pr ofile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wi ld-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However. macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild- type and mutant channels. Concatenation of the CT domain of Cx43 to amino a cids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance a nd voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state in volves a ball-and-chain type of interaction between the CT domain and the p ore-forming region. This interaction can be either homologous (Cx40 truncat ion with Cx40CT) or heterologous (with the Cx43CT).