Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.     

Different domains are critical for oligomerization compatibility of different connexins

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.     

Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants.

The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.     

Differential Oligomerization of Endoplasmic Reticulum-Retained Connexin43/Connexin32 Chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization

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.     

A carboxyl terminal domain of connexin43 is critical for gap junction plaque formation but not for homo- or hetero-oligomerization

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.     

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

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.     

Functional analysis of selective interactions among rodent connexins.

One consequence of the diversity in gap junction structural proteins is that cells expressing different connexins may come into contact and form intercellular channels that are mixed in connexin content. We have systematically examined the ability of adjacent cells expressing different connexins to communicate, and found that all connexins exhibit specificity in their interactions. Two extreme examples of selectivity were observed. Connexin40 (Cx40) was highly restricted in its ability to make heterotypic channels, functionally interacting with Cx37, but failing to do so when paired with Cx26, Cx32, Cx43, Cx46, and Cx50. In contrast, Cx46 interacted well with all connexins tested except Cx40. To explore the molecular basis of connexin compatibility and voltage gating, we utilized a chimera consisting of Cx32 from the N-terminus to the second transmembrane domain, fused to Cx43 from the middle cytoplasmic loop to the C-terminus. The chimeric connexin behaved like Cx43 with regard to selectivity and like Cx32 with regard to voltage dependence. Taken together, these results demonstrate that the second but not the first extracellular domain affects compatibility, whereas voltage gating is strongly influenced by sequences between the N-terminus and the second transmembrane domain.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Age-related alteration of gap junction distribution and connexin expression in rat aortic endothelium.

We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and > or =20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week (p<0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until > or =20 months of age (> or =20 months vs. 28 days, p<0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages (p<0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.     

Regulation of Connexin43 Oligomerization is Saturable

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.     

Regulation of connexin43 oligomerization is saturable

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Heteromeric mixing of connexins: compatibility of partners and functional consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.     

Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins.

The cellular distribution of connexin40 (Cx40), a newly cloned gap junction structural protein, was examined by immunofluorescence microscopy using two different specific anti-peptide antibodies. Cx40 was detected in the endothelium of muscular as well as elastic arteries in a punctate pattern consistent with the known distribution of gap junctions. However, it was not detected in other cells of the vascular wall. By contrast, Cx43, another connexin present in the cardiovascular system, was not detected in endothelial cells of muscular arteries but was abundant in the myocardium and aortic smooth muscle. We have tested the ability of these connexins to interact functionally. Cx40 was functionally expressed in pairs of Xenopus oocytes and induced the formation of intercellular channels with unique voltage dependence. Unexpectedly, communication did not occur when oocytes expressing Cx40 were paired with those expressing Cx43, although each could interact with a different connexin, Cx37, to form gap junction channels in paired oocytes. These findings indicate that establishment of intercellular communication can be spatially regulated by the selective expression of different connexins and suggest a mechanism that may operate to control the extent of communication between cells.     

Heteromeric Mixing of Connexins: Compatibility of Partners and Functional Consequences

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.