Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia

Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43. Together the fluorescence-based assay was combined with patch clamp analysis to assess the dominant negative potency of Cx43 mutants. Our results revealed that the ODDD-linked Cx43 mutants, G21R and G138R, as well as amino terminus green fluorescent protein-tagged Cx43, were able to co-localize with wild-type Cx43 at the gap junction plaque-like structures and to co-immunoprecipitate with wild-type Cx43. All Cx43 mutants demonstrated dominant negative action on gap junctional conductance of wild-type Cx43 but not that of Cx32. More interestingly, these Cx43 mutants demonstrated different potencies in inhibiting the function of wild-type Cx43 with the G21R mutant being two times more potent than the G138R mutant. The potency difference in the dominant negative properties of ODDD-linked Cx43 mutants may have clinical implications for the various symptoms and disease severity observed in ODDD patients.     

Functional Characterization of Oculodentodigital Dysplasia-Associated Cx43 Mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

Mutations in the second extracellular region of connexin 43 prevent localization to the plasma membrane,but do not affect its ability to suppress cell growth

Connexin 43 (Cx43), the most widely expressed gap junction protein, has a role in regulation of cell growth. In this study, we demonstrate that the point mutations F199L, R202E, and E205R in the second extracellular region of Cx43 prevent localization of the mutant proteins to the plasma membrane. The mutants were aberrantly localized in the cytoplasm if expressed in HeLa cells, which lack Cx43. Coexpression with wild-type Cx43 promoted localization of the F199L and R202E mutant proteins to the plasma membrane. By dye transfer assay, we showed that gap junctional intercellular communication (GJC) is decreased in cells expressing the mutants, compared to Cx43 wild-type-expressing cells. However, the F199L mutant does not appear to have a dominant-negative effect on GJC. Despite the loss of GJC, the ability of the F199L Cx43 mutant to inhibit growth of either Cx43-/- cells or two cancer cell lines, HeLa and C6 glioma cells, was similar to that of the wild-type Cx43. In addition, we showed that both R202E and E205R Cx43 mutant expressions cause growth retardation of HeLa cells. Therefore, the point mutations in the second extracellular region of Cx43 do not affect the ability of the mutant proteins in vitro to suppress cell growth, although they prevent localization to the plasma membrane. The results support the concept that regulation of cell growth by Cx43 does not necessarily require GJC and suggest that the growth-suppressive properties of Cx43 may be independent of the second extracellular loop.     

Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.     

Some Oculodentodigital Dysplasia-Associated Cx43 Mutations Cause Increased Hemichannel Activity in Addition to Deficient Gap Junction Channels

Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in ODDD patients who are deficient in Cx43 gap junction channels. Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Heteromeric connexons formed by the lens connexins, connexin43 and connexin56

In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.     

Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

Lysosomal and proteasomal degradation play distinct roles in the life cycle of Cx43 in gap junctional intercellular communication-deficient and -competent breast tumor cells

The present study was designed to determine the specific roles played by lysosomes and proteasomes in the degradation of Cx43 in both gap junctional intercellular communication-deficient MDA-MB-231 and -competent BICR-M1Rk cells. In MDA-MB-231 cells, immunolocalization and brefeldin A protein transport blocking studies revealed that there was a propensity for newly synthesized Cx43 to be transported to lysosomes. On the other hand, light and electron microscopic analysis of BICR-M1Rk cells showed that Cx43 gap junctions were prevalent with a subpopulation of intracellular Cx43 localized to lysosomes. In both cell types, Western blots revealed a notable increase in total cellular Cx43 in response to lysosome inhibitors. Interestingly, lactacystin inhibition of proteosomal degradation in MDA-MB-231 cells resulted in a marked increase in phosphorylated Cx43 at the expense of non-phosphorylated Cx43, and this change corresponded with an increase in "oversized" gap junction plaques. In BICR-M1Rk cells, lactacystin treatment partially prevented the BFA-induced loss of gap junctions. Together, our data suggests that lysosomes play a key role in not only degrading internalized gap junction in BICR-M1Rk cells but also in degrading Cx43 delivered from early secretory compartments to lysosomes in MDA-MB-231 cells. Overall proteasomal degradation regulates the stability of phosphorylated Cx43 and appears to promote the internalization of Cx43 from the cell surface.     

Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.     

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.     

Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its lysosomal degradation

Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts.     

A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes

Dominant mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause oculodentodigital dysplasia (ODDD), a syndrome affecting multiple tissues, including the central nervous system (CNS). We investigated the effects of the G60S mutant, which causes a similar, dominant phenotype in mice (Gja1(Jrt/+)). Astrocytes in acute brain slices from Gja1(Jrt/+) mice transfer sulforhodamine-B comparably to that in their wild-type (WT) littermates. Further, astrocytes and cardiomyocytes cultured from Gja1(Jrt/+) mice showed a comparable transfer of lucifer yellow to those from WT mice. In transfected cells, the G60S mutant formed gap junction (GJ) plaques but not functional channels. In co-transfected cells, the G60S mutant co-immunoprecipitated with WT Cx43, but did not diminish GJ coupling as measured by dual patch clamp. Thus, whereas G60S has dominant effects, it did not appreciably reduce GJ coupling.     

Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant-negative effect on the wild-type protein in HeLa cells

To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.