Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32

Abstract

The gene family encoding gap junction proteins (connexins) consists of several known members, and multiple connexins are frequently coexpressed by coupled cells. To characterize the channel properties of the major rat liver gap junction protein (connexin 32) in isolation from other gap junction proteins, we have introduced the cDNA encoding it into a human hepatoma cell line (SKHep1) in which we have identified a gap junction deficiency. In this cell line, dye coupling was absent and junctional conductance was near zero. Connexins and connexin 32 mRNA were not detectable by immunocytochemistry and Northern blot analysis. After transfection and selection, cells were strongly coupled with regard to dye and electrical current, and connexin 32 mRNA and punctate connexin 32-immunoreactive membrane contacts were abundant. Functional gap junction channels were still expressed after 19 passages of the cells, indicating stable transfection. When junctional conductance was rendered reversibly low by exposing the cells to agents that uncouple other cell types, currents through single gap junction channels could be observed. The unitary conductance of these expressed channels was about 120-150 pS, a value that is distinctly larger than in heart cells, which express a different gap junction protein.