Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manif ests altered trafficking of various plasma membrane proteins. In particular , there is a striking loss of State 2 asialoglycoprotein receptors. This ce ll line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expressio n or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 rev ealed that fewer and smaller gap junctions were present at appositional mem brane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication betwe en Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca2+ waves. Isoelectric fo cusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cel l line. The effects of the Trf1 mutation on assembly and function of gap ju nctions indicate that this mutation influences trafficking of Cx43, Connexi ns differ in several respects from other membrane proteins thus far analyze d in Trf1 mutants: gap junctions localize exclusively to the lateral cell s urface; they are not glycoproteins; and they do not play a role in endocyti c pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the traffickin g pathway of cell-surface proteins, irrespective of their ultimate destinat ion on the cell surface or their glycosylation profile.