Functional characteristics of heterogeneous Cx40/Cx43 gap junction channel formation

Abstract

Cells of the cardiovascular system express multiple connexins (Cx) with Cx40 and Cx43 being commonly coexpressed in many tissues. The expression levels of connexins are dynamic and can vary in response to a growth stimulus. It is not clear why cells express multiple connexins, or what advantage such dynamic regulation of expression patterns have on cell function. These issues are further complicated by the ability of some connexins to interact to form heterogeneous gap junction channels, with little being known regarding functional properties of such channels. The purpose of these experiments was threefold: (1) To determine whether Cx40 and Cx43 are capable of interacting to form heteromeric/heterotypic gap junction channels; (2) To characterize the functional properties of Cx40/Cx43 heteromeric/heterotypic channels; and (3) To determine the effect that changing Cx40:Cx43 expression ratio has on functional properties of heteromeric/heterotypic channels. Cell lines were developed that express only Cx43 (Rin43), Cx40 (Rin40), and Cx40 and Cx43 in varying Cx40:Cx43 expression ratios (6B5n, A7r5, A7r540C1, and A7r540C3). The Cx40:Cx43 expression ratios in the 6B5N, A7r5, A7r540C1, and A7r540C3 cells are approximately 1:1, 3:1, 5:1, and 10:1, respectively. Functional properties of the gap junction channels formed between these cells were determined using both electrophysiological and dye coupling techniques. Pairing of Rin43 and Rin40 cells demonstrated that Cx40 and Cx43 are capable of forming homomeric/heterotypic gap junctions with unique voltage-dependent gating and single channel behaviors. Rin43/A7r5 cell pairs displayed voltage-dependent gating and single channel conductance profiles that could only be explained by the presence of heteromeric/heterotypic gap junction channels between these cells. Pairing Rin43 cells with coexpressing cells of high Cx40:Cx43 expression ratio resulted in channel activities that were not predicted by the gating and conductance patterns of Cx40/Cx43 heterotypic channels. However, the dye coupling characteristics of these same cells in coculture demonstrated that the permeability of the channels formed between these cell types reflected that of Cx40 channels. In summary, Cx40 and Cx43 are capable of forming heteromeric/heterotypic gap junction channels. Increasing the Cx40:Cx43 ratio in coexpressing cells results in channels with unique gating and conductance properties, however dye permeability of these cells is predicted by their relative Cx40 content. Therefore, varying Cx40:Cx43 expression ratio provides cells with a mechanism to finely control the types of molecules shared between cells.