Xiang-Qun Gong

Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins

Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional intercellular communication. Localization of exogenously expressed pannexin 1 (Panx1) and pannexin 3 (Panx3), together with pharmacological studies, revealed a cell surface distribution profile and life cycle dynamics that were distinct from connexin 43 (Cx43, encoded by Gja1). Furthermore, N-glycosidase treatment showed that both Panx1 (∼41-48 kD species) and Panx3 (∼43 kD) were glycosylated, whereas N-linked glycosylation-defective mutants exhibited a decreased ability to be transported to the cell surface. Tissue surveys revealed the expression of Panx1 in several murine tissues – including in cartilage, skin, spleen and brain – whereas Panx3 expression was prevalent in skin and cartilage with a second higher-molecular-weight species present in a broad range of tissues. Tissue-specific localization patterns of Panx1 and Panx3 ranging from distinct cell surface clusters to intracellular profiles were revealed by immunostaining of skin and spleen sections. Finally, functional assays in cultured cells transiently expressing Panx1 and Panx3 were incapable of forming intercellular channels, but assembled into functional cell surface channels. Collectively, these studies show that Panx1 and Panx3 have many characteristics that are distinct from Cx43 and that these proteins probably play an important biological role as single membrane channels.

A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

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 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.

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.

Fate of connexin43 in cardiac tissue harbouring a disease-linked connexin43 mutant

AIMS More than 40 mutations in the GJA1 gene encoding connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD), a pleiotropic, autosomal dominant disorder. We hypothesized that even with a significant reduction in the levels of Cx43 in a mutant mouse model of ODDD (Gja1(Jrt/+)) harbouring a G60S mutation (Cx43(G60S)), cardiomyocyte function may only be moderately compromised given that a majority of mutant mice typically survive. METHODS AND RESULTS Western blotting and quantitative reverse transcriptase-polymerase chain reaction in conjunction with immunofluorescence were used to assess the expression and localization of Cx43 in hearts and cultured cardiomyocytes from wild-type and Gja1(Jrt/+) mice. Dye-coupling and dual whole cell patch-clamp recordings were also used to assess the gap junction channel status in cultured cardiomyocytes from wild-type and mutant mice. Cardiac tissue from adult Gja1(Jrt/+) mice revealed a 60-80% reduction in Cx43 protein with a preferential loss of the highly phosphorylated forms of Cx43. Compensation via the up-regulation of Cx40 or Cx45 was not observed. Immunofluorescent analysis of cultured cardiomyocytes revealed a trafficking defect, with a decrease in Cx43 plaques and a large population of Cx43 being retained in the Golgi apparatus. However, cultured cardiomyocytes from mutant mice remained beating with a 50% decrease in coupling conductance. CONCLUSION These results suggest that the Cx43(G60S) mutant impairs normal trafficking and function of co-expressed Cx43 with no dramatic effect on cardiomyocyte function, suggesting that Cx43 is biosynthesized in excess of an essential need.

Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn175 of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

D‐Aspartate and NMDA, but not L‐aspartate, block AMPA receptors in rat hippocampal neurons

1 The amino acid, D‐aspartate, exists in the mammalian brain and is an agonist at the N‐methyl‐D‐aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D‐aspartate on α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazolepropionate receptors (AMPARs) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D‐aspartate inhibited kainate‐induced AMPAR current in hippocampal neurons. The inhibitory action of D‐aspartate on kainate‐induced AMPAR current was concentration‐dependent and was voltage‐independent in the tested voltage range (−80 to +60 mV). 3 The estimated EC50 of the L‐glutamate‐induced AMPAR current was increased in the presence of D‐aspartate, while the estimated maximum L‐glutamate‐induced AMPAR current was not changed. D‐aspartate concentration‐dependently shifted the dose–response curve of kainate to the right. Schild plot analysis indicated that D‐aspartate acts competitively to block AMPARs. The Kb for D‐aspartate was estimated to be 0.93 mM. 4 D‐Aspartate also blocked L‐glutamate‐induced current in Xenopus laevis oocytes that expressed recombinant homomeric AMPARs. 5 NMDA possessed similar inhibitory action on AMPARs. However, L‐aspartate had little inhibitory action on AMPARs. 6 D‐Aspartate, but not L‐aspartate, was found to reduce the amplitude of miniature excitatory postsynaptic current in cultured hippocampal neurons. 7 Our data are consistent with a model in which D‐aspartate directly competes with kainate and L‐glutamate in binding to the agonist binding site of AMPARs. The prevalence of D‐aspartate in the brain suggests a possible role of D‐aspartate in modulating AMPAR‐mediated fast excitatory synaptic transmission.

Structure and Functional Studies of N-terminal Cx43 Mutants Linked to Oculodentodigital Dysplasia

Mutations in the connexin-43 gap junction protein cause the developmental disease known as oculodentodigital dysplasia. Structure and function approaches are used to demonstrate that the nature of the missense mutation in the amino-terminal domain of connexin-43 governs the mechanism that leads to loss of connexin-43 function.

The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients.