0123 : Effect of the connexin43 downregulation on the electrical gap junction channels in RLE cells

Abstract

In cells that co-express connexin 43 (Cx43) and Cx40 we have previously shown that the formation and the electrical properties of the gap junction channels (GJCs) is regulated by the increased expression of Cx40 and the ratio of Cx43:Cx40, which demonstrated a relative contribution of each Cx. We aim to further investigate this regulation in the case of a down-regulation of the expression of Cx43. Rat Liver Epithelial (RLE) cells that express Cx43 have been stably transfected with the inducible Ecdysone system to co-express accurate ratios of Cx43:Cx40 (0.9, 1.0 and 1.8). The level of expression of Cx40 is controlled by different concentrations of Ponasterone A, while the level of expression of Cx43 is constant. Cells are transfected with specific small interference RNA (SiRNA) against Cx43 to investigate the specific contribution of Cx43 in the formation and the electrical properties of GJCs by performing dual voltage clamp recordings on cell pairs. Unexpectedly, in native cells the down-regulation of Cx43 increased the electrical coupling, without affecting the voltage-dependence and the single channel conductances characteristic of homotypic Cx43 GJCs. On the contrary, in Cx40-induced cells the down-regulation of Cx43 increased the electrical coupling, the voltage-dependence and the unitary conductance of GJCs in a ratio dependent manner. This suggests a different hetero-assembling of Cxs to non-treated cells, while none homotypic Cx40 channels were observed. These data reveals a specific homo- and hetero-assembling of Cxs and regulation of the formation of GJCs ensured by distinct contribution of Cx43 and Cx40. This leads to determinant specific electrical properties of GJCs for regulating the impulse propagation in the healthy heart, which become pro-arrhythmic in the case of Cxs dysfunction. The biochemical characterization of the down-regulation of Cx43 will be correlated to our findings and to the cardiac pattern of expression. The author hereby declares no conflict of interest