Targeted Gap Junction Protein Constructs Reveal Connexin-specific Differences in Oligomerization

Abstract

To define further the mechanisms of gap junction protein (connexin (Cx)) oligomerization without pharmacologic disruption, we have examined the transport and assembly of connexin constructs containing C-terminal di-lysine-based endoplasmic reticulum (ER) (HKKSL) or ER-Golgi intermediate compartment (AKKFF) targeting sequences. By immunofluorescence microscopy, Cx43-HKKSL transiently transfected into HeLa cells showed a predominantly ER localization, although Cx43-AKKFF was localized to the perinuclear region of the cell. Sucrose gradient analysis of Triton X-100-solubilized connexins showed that either Cx43-HKKSL or Cx43-AKKFF expressed alone by HeLa cells was maintained as an apparent monomer. In contrast to Cx43-HKKSL, Cx32-HKKSL was maintained in the ER as stable hexamers, consistent with the notion that Cx32 and Cx43 oligomerization occur in distinct intracellular compartments. Furthermore, Cx43-HKKSL and Cx43-AKKFF inhibited trafficking of Cx43 and Cx46 to the plasma membrane. The inhibitory effect was because of the formation of mixed oligomers between Cx43-HKKSL or Cx43-AKKF and wild type Cx43 or Cx46. Taken together, these results suggest that Cx43-HKKSL and Cx43-AKKFF recirculate through compartments where oligomerization occurs and may be maintained as apparent monomers by a putative Cx43-specific quality control mechanism.

Identification of rab20 as a Potential Regulator of Connexin43 Trafficking

“…However, added expression of Cx43 alone did not have the capacity to compensate for Cx43-HKKSL overexpression, since 3(3) cells transfected with myctagged Cx43 continued to retain Cx43-HKKSL in the perinuclear region of the cells (Figure 3c to e). Although this is subject to the caveat that we tested this effect using myc-tagged Cx43, Cx43-HKKSL has also been shown to bind and restrict the transport of untagged Cx46 (Das Sarma et al 2002).…”

“…Stably transfected 1(6) and 3(3) HeLa/Cx43-HKKSL cell lines were produced and characterized as previously described (Das Sarma et al 2002Maza et al 2003Maza et al , 2005. Rabbit anti-Cx43 was from Sigma (St. Louis, MO), anti-EGFP (enhanced green florescent protein) was from Invitrogen (Carlsbad, CA).…”

“…myc-tagged connexin43 was produced as described (Das Sarma et al 2002). rab20 cDNA was amplified from the library using High Fidelity DNA polymerase (Roche Diagnostics) with 5 -CCCAAGCTTA CGGGAAGATG AGGAAG CCC-3 and 5 -GCTCTAGACC TCGAAAGTCA GGCACAAC-3 as primers.…”

“…As a platform for this assay, we used HeLa cells that were stably transfected with a Cx43 construct containing an endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) (Das Sarma et al 2002Maza et al 2003Maza et al , 2005. Most of these transfectants retained Cx43-HKKSL in the ER, as demonstrated by immunofluorescence microscopy (see Figure 1).…”

A Carboxyl Terminal Domain of Connexin43 Is Critical for Gap Junction Plaque Formation but not for Homo- or Hetero-Oligomerization

“…Oligomerization may involve all identical subunits (to form homomeric connexons) or various combinations of compatible connexins (to form heteromeric connexons). The cellular process of connexin oligomerization is beginning to be elucidated (1,6,8,18). We and others have identified a number of compatible or incompatible partners for the formation of heteromeric connexons (3,4,5,10,11,15).…”