Cx29 and Cx32, two connexins expressed by myelinating glia, do not interact and are functionally distinct

Abstract

In rodents, oligodendrocytes and myelinating Schwann cells express connexin32 (Cx32) and Cx29, which have different localizations in the two cell types. We show here that, in contrast to Cx32, Cx29 does not form gap junction plaques or functional gap junctions in transfected cells. Furthermore, when expressed together, Cx29 and Cx32 are not colocalized and do not coimmunoprecipitate. To determine the structural basis of their divergent behavior, we generated a series of chimeric Cx32-Cx29 proteins by exchanging their intracellular loops and/or their C-terminal cytoplasmic tails. Although some chimerae reach the cell membrane, others appear to be largely localized intracellularly; none form gap junction plaques or functional gap junctions. Substituting the C-terminus or the intracellular loop and the C-terminus of Cx32 with those of Cx29 does not disrupt their colocalization or coimmunoprecipitation with Cx32. Substituting the C-terminus of Cx29 with that of Cx32 does not disrupt the coimmunoprecipitation or the colocalization with Cx29, whereas substituting both the intracellular loop and the C-terminus of Cx32 with those of Cx29 diminishes the coimmunoprecipitation with Cx29. Conversely, the Cx32 chimera that contains the intracellular loop of Cx29 coimmunoprecipitates with Cx29, indicating that the intracellular loop participates in Cx29-Cx29 interactions. These data indicate that homomeric interactions of Cx29 and especially Cx32 largely require other domains: the N-terminus, transmembrane domains, and extracellular loops. Substituting the intracellular loop and/or tail of Cx32 with those of Cx29 appears to prevent Cx32 from forming functional gap junctions.

Fig. 1

Localization of Cx29, Cx32, and Cx29/Cx32 chimerae. The upper panels depict the structures of mouse Cx29, mouse Cx32, and the Cx29/Cx32 chimerae in which the cytoplasmic loop (L) and/or C-terminus (T) were exchanged. A–H are deconvolved images of transiently transfectedHeLa cells that express the indicated construct, immunostained with a rabbit antiserum against the C-terminus of Cx29 (RbαCx29T; Altevogt et al., 2002) or Cx32 (Rbα32T; Chemicon) ormonoclonal antibodies against the intracellular loop (MαCx32L; Zymed) or C-terminus (MαCx32T; 7C6.C7) of Cx32 as indicated, and counterstained with DAPI (blue). Cx29 (A), Cx29L³² (B), Cx29T³² (C), and Cx29L³²T³² (D) are localized to the cell membrane, Cx32 (E) is localized to gap junction plaques (arrowheads) between apposed cell membranes; Cx32L²⁹ (F) shows diffuse intracellular staining, and Cx32T²⁹ (G) andCx32L²⁹T²⁹ (H) are foundmostly in intracellular puncta. Scale bar = 10 μm.

Fig. 2

A–H: Cx29 or Cx29/Cx32 chimerae do not form functional gap junctions. These are digital images of scrape-loaded, confluent plates of bulk-selected HeLa cells that stably express the indicated constructs. The cells were incubated in 1% LY and imaged 5 min after injury with fluorescence optics to visualize LY. In cells expressing Cx32, LY diffuses into adjacent cells, whereas, in cells expressing Cx29 or one of the chimeric constructs, LY only labeled injured cells. Scale bar = 20 μm.

Fig. 3

FRAP analysis of HeLa cells that stably express Cx29. The upper panels are digital images of parental HeLa cells or cloned HeLa cells that stably express mouse Cx29 or human Cx47. The cells were preloaded with calcein, and a small group of cells in the middle of the image was photobleached. Images are shown from cells 5 sec before (−5 sec) and 5 sec (+5 sec) and 1,000 sec (+1,000 sec) after photobleaching. The lower panel is a quantitative analysis of FRAP. Fluorescence was measured in the bleached area every 10 sec for 30 min. For every experiment, the measured region was considered to be 100% prior to bleaching, and 0% immediately following bleaching. Note the recovery of fluorescence in cells that express Cx47, but not in parental cells or in cells that express Cx29. The error bars show SEM, which is too small to be illustrated for many samples. The number of imaged fields is as follows: HeLa cells (n = 8); HeLa cells that stably express mouse Cx29 (n = 11); HeLa cells that stably human Cx47 (n = 11). Scale bar = 20 μm.

Fig. 4

Immunoblot analysis of Cx29, Cx32, and Cx29/Cx32 chimerae. These are immunoblots of three identical membranes from lysates of HeLa cells that were transiently transfected with the indicated constructs; 80 μg of each sample were separated by SDS-PAGE, transferred to a membrane, and probed with mouse monoclonal antibodies against the intracellular loop (Cx32L; Zymed) or Cterminus (Cx32T; 7C6.C7) of Cx32, or a rabbit antiserum against the C-terminus of Cx29 (Cx29T; Zymed). The membranes were incubated with peroxidase-conjugated donkey antibodies against mouse or rabbit IgG and developed with ECL. Although the intensities of the bands vary for each construct, Cx32L²⁹ (lane 5) and Cx32L²⁹T²⁹ (lane 3) being the lightest, monomers (arrowheads) of the appropriate size are detected with the appropriate antibodies; the higher molecular mass bands in some lanes are dimers (double arrowheads). The asterisk marks a spurious spot in the Cx32T²⁹ sample (lane 7). The image of the Coomassie-stained gel is shown to document equal loading of the samples.

Fig. 5

Cx29, Cx32, and some Cx29/Cx32 chimerae reach the cell membrane. HeLa cells were transiently transfected with the indicated constructs, then biotinylated, washed, and lysed. The lysates were incubated with avidin beads, and the avidin-bound fractions and 25 μl of unbound fractions were separated on SDS-PAGE gels and transferred to two membranes, which were hybridized with either a rabbit antiserum against the C-terminus of Cx29 (Cx29T; Zymed) or a mouse monoclonal antibody against the C-terminus of Cx32 (Cx32T; Cx32T). The membranes were incubated with peroxidase-conjugated donkey antibodies against rabbit or mouse IgG and developed with ECL. A: Two exposure times are shown; monomer (arrowhead), dimers (double arrowhead), and trimers (triple arrowheads) are seen in the bound (and unbound) fraction from biotinylated cells that express Cx29 (lane 9), Cx32 (lane 18), Cx29L³² (lane 7), Cx29T³² (lane 16), and Cx29L³²T³² (lane 14). Weaker signal is detected in the bound and unbound fractions for Cx32T²⁹ (lane 5) and Cx32L²⁹T²⁹ (lane 3), and no signal is detected for Cx32L²⁹ (lane 12). B: The membranes were stripped and reprobed with a rabbit antiserum against actin. Note that all unbound fractions contain a more robust actin signal (arrows) than do their corresponding bound samples, demonstrating that intracellular proteins were not biotinylated. The other bands in the left image result from the incompletely stripping of the rabbit antiserum against Cx29 C-terminus. The images of the Coomassie-stained gels document the loading of the samples (C).

Fig. 6

Coimmunoprecipation of Cx29, Cx32, and selected chimerae. Cells cotransfected with the indicated constructs and lysates were immunoprecipitated with a rabbit antiserum against the C-terminus of Cx29 (Zymed), the immunoprecipitates were separated on three gels, transferred to membranes, which were hybridized together with a mouse monoclonal antibody against the C-terminus of Cx32 (Cx32T; 7C6.C7) and developed with ECL. The blots were rehybridized together with an antiserum against the C-terminus of Cx29 (Cx29T; Zymed) and developed with ECL. In each row, the film was exposed for the same amount of time, except for top right panel, which was exposed longer (3 min). Note that Cx32 is not coimmunoprecipitated with Cx29 (lane 3); that replacing the C-terminus (Cx32T²⁹; lane 6) or even the C-terminus and the intracellular loop of Cx32 (Cx32L²⁹T²⁹; lane 9) with those of Cx29 does not prevent the coimmunoprecipitation with Cx32; that replacing the C-terminus and the intracellular loop of Cx29 (Cx29L³²T³²; lane 15) with those of Cx32 diminished the coimmunoprecipitation, with Cx29 more than replacing the C-terminus alone (Cx29T³²; lane 12). Furthermore, replacing the intracellular loop of Cx32 with that of Cx29 (Cx32L²⁹) promotes an interaction with Cx29 (lane 21), whereas replacing the intracellular loop of Cx29 with that of Cx32 (Cx29L³²) does not promote an interaction with Cx32 (lane 18). The arrow marks a spurious band that is present in all samples in the lower panels. The single, double, and triple arrowheads indicate the positions of connexin monomers, dimers, and multimers, respectively.

Fig. 7

Localization of Cx29, Cx32, and various chimerae in transfected cells. These are images of HeLa cells that were transiently transfected to coexpress the indicated constructs and immunostained with a rabbit antiserum against the C-terminus of Cx29 (Cx29T; Altevogt et al. 2002) and a monoclonal antibody against the C-terminus of Cx32 (Cx32T; 7C6.C7). Note that Cx32 forms gap junction plaques (arrowheads), but neither Cx32 and Cx29 (A) nor Cx32 and Cx29L³² (G) appear to be colocalized; that Cx29 and Cx29T³² (B) as well as Cx29 and Cx29L³²T³² (C) are colocalized throughout the cell membrane; that the Cx29 that is localized to the cell membrane does not “mislocalize” Cx32L²⁹, which is localized in the ER (D); and that Cx32 and Cx32T²⁹ (E) as well as Cx32 and Cx32L²⁹T²⁹ (F) are colocalized in intracellular puncta. Scale bar ~10 μm.